Methods for diagnosing, prognosing, and treating colorectal cancer using biomarker expression

ABSTRACT

Dysregulated expression of microRNAs (miRNAs) has emerged as a hallmark feature in human cancers. Aspects of the disclosure relate to methods for selecting optimal therapy for a patient from several alternative treatment options. A major clinical challenge in cancer treatment is to identify the subset of patients who will benefit from a therapeutic regimen, both in metastatic and adjuvant settings. The number of anti-cancer drugs and multi-drug combinations has increased substantially in the past decade, however, treatments continue to be applied empirically using a trial-and-error approach. Here methods and compositions are provided to determine the optimal treatment option for cancer patients.

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/464,781, filed Feb. 28, 2017, herebyincorporated by reference in its entirety.

This invention was made with government support under Grant Nos.R01CA202797, U01CA187956, R01CA184792, R01CA072851 and R01CA181572awarded by the National Cancer Institute, National Institutes of Health.The government has certain rights in the invention.

BACKGROUND 1. Field of the Invention

The present invention relates generally to the fields of molecularbiology and oncology. More particularly, it concerns methods andcompositions involving cancer prognosis, diagnosis, and treatment.

2. Description of Related Art

Colorectal cancer (CRC) is one of the most frequently diagnosedmalignancies and a leading cause of cancer-related deaths worldwide.High degree of mortality associated with CRC is largely due to latedisease detection and lack of availability of adequate prognosticbiomarkers, including the currently used tumor-node-metastasis (TNM)classification system from the American Joint Committee on Cancer forpredicting tumor prognosis and recurrence. This highlights the need todevelop robust prognostic biomarkers for CRC, and the expectations arethat such biomarkers must offer a superior prognostic clinicalusefulness compared to existing TNM staging classification. In addition,such biomarkers must perform independent of the existing classificationcriteria, and possess adequate prognostic significance for specificsubgroups defined by node-negative (stage II) or node-positive (stageIII) CRC patients.

SUMMARY OF THE DISCLOSURE

The current disclosure fulfills a need in the art by providing moreeffective therapeutic treatments and diagnostic/prognostic methods forcolorectal cancer based on the expression or activity level ofbiomarkers. Aspects of the disclosure relate to a method for treating apatient determined to have colorectal cancer comprising: administeringadjuvant therapy to the patient; wherein the patient was determined tohave one or more of the following: differential expression of one ormore miRNA, lncRNA, piRNA, mRNA, protein, or 5hmC DNA-modified genebiomarkers compared to a control sample, wherein the one or morebiomarkers are selected from: i) differential expression of miR-30b,miR-32, miR-33a, miR-34a, miR-101, miR-181b, miR-188, miR-191, miR-193b,miR-195, miR-200, miR-200b, miR-362, miR-409, miR-424, miR-425, miR-429,miR-432, miR-592, miR-744, miR-758, miR-1246, miR-3182, miR-3605,miR-3677, miR-4284, miR-4326, CCAT1, CCAT2; piRNA: DQ596309, DQ570994(piRACC), DQ595807 (piR61919), piR30652 (DQ570540), and/or piR31111(DQ570999); ii) differential levels of 5hmC DNA modification of genes:P2RX4, CRISPLD2, and/or FKBP4; and iii) differential mRNA geneexpression and/or protein level of: CD44v6, AMT, C2CD4A, CYP2B6, DEFA6,FOXA1, GSTT1, LYZ, MGAT5, MMP1, MMP9, NOS2, PIGR, PRAC1, RPL39L, RCC1,SPAG16, AMT2, and/or ITGBL1. In some embodiments, the subject wasdetermined to have differential expression of at least, at most, orexactly one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, or twenty (or any derivable range therein) of thebiomarkers described herein.

In some embodiments, the expression level of lncRNAs: CCAT1 and/or CCAT2were increased compared to the control; the expression level of piRNAs:DQ596309, DQ570994 (piRACC), DQ595807, DQ570540, and/or DQ570999 wereincreased compared to the control; and/or the level of 5hmC of genes:P2RX4, CRISPLD2, and/or FKBP4 is decreased compared to the control. Insome embodiments, the expression level of miR-30b, miR-32, miR-33a,miR-34a, miR-101, miR-181b, miR-188, miR-191, miR-193b, miR-195,miR-200, miR-200b, miR-362, miR-409, miR-424, miR-425, miR-429, miR-432,miR-592, miR-744, miR-758, miR-1246, miR-3182, miR-3605, miR-3677,miR-4284, and/or -4326 were increased compared to the control. In someembodiments, miR-30b, miR-32, miR-33a, miR-34a, miR-101, miR-181b,miR-188, miR-191, miR-193b, miR-195, miR-200, miR-200b, miR-362,miR-409, miR-424, miR-425, miR-429, miR-432, miR-592, miR-744, miR-758,miR-1246, miR-3182, miR-3605, miR-3677, miR-4284, and/or miR-4326 weredecreased compared to the control. In some embodiments, CD44v6, AMT,C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1, LYZ, MGAT5, MMP1, MMP9, NOS2, PIGR,PRAC1, RPL39L, RCC1, SPAG16, AMT2, and/or ITGBL1 were increased comparedto control. In some embodiments, CD44v6, AMT, C2CD4A, CYP2B6, DEFA6,FOXA1, GSTT1, LYZ, MGAT5, MMP1, MMP9, NOS2, PIGR, PRAC1, RPL39L, RCC1,SPAG16, AMT2, and/or ITGBL1 were decreased compared to control.

A further aspect of the disclosure relates to a method for determiningwhether a patient diagnosed with Stage I, II, III, or IV colorectalcancer is high or low risk, wherein the method comprises: determiningthat the patient is high risk when one or more miRNA, lncRNA, piRNA,mRNA, protein, or 5hmC modified gene biomarkers are determined to bedifferentially expressed in a biological sample from the patientcompared to a control; or determining that the patient is low risk whenone or more of a miRNA, lncRNA, piRNA, mRNA, protein, or 5hmC modifiedgene biomarkers are determined to be not significantly different inexpression in a biological sample from the patient compared to acontrol; wherein the one or more biomarkers are selected from: i)differential expression of miR-30b, miR-32, miR-33a, miR-34a, miR-101,miR-181b, miR-188, miR-191, miR-193b, miR-195, miR-200, miR-200b,miR-362, miR-409, miR-424, miR-425, miR-429, miR-432, miR-592, miR-744,miR-758, miR-1246, miR-3182, miR-3605, miR-3677, miR-4284, miR-4326,CCAT1, CCAT2; piRNA: DQ596309, DQ570994 (piRACC), DQ595807, DQ570540,and/or DQ570999; ii) differential 5hmC DNA modification levels of genes:P2RX4, CRISPLD2, and/or FKBP4; and iii) differential mRNA geneexpression and/or protein level of: CD44v6, AMT, C2CD4A, CYP2B6, DEFA6,FOXA1, GSTT1, LYZ, MGAT5, MMP1, MMP9, NOS2, PIGR, PRAC1, RPL39L, RCC1,SPAG16, AMT2, and/or ITGBL1.

In some embodiments, the patient is determined as high risk when theexpression level of lncRNAs: CCAT1 and/or CCAT2 were determined to beincreased compared to the control; the expression level of piRNAs:DQ596309, DQ570994 (piRACC), DQ595807, DQ570540, and/or DQ570999 weredetermined to be increased compared to the control; and/or the level of5hmC of genes: P2RX4, CRISPLD2, and/or FKBP4 were determined to bedecreased compared to the control. In some embodiments, the patient isdetermined to be high risk when the expression level of miR-30b, miR-32,miR-33a, miR-34a, miR-101, miR-181b, miR-188, miR-191, miR-193b,miR-195, miR-200, miR-200b, miR-362, miR-409, miR-424, miR-425, miR-429,miR-432, miR-592, miR-744, miR-758, miR-1246, miR-3182, miR-3605,miR-3677, miR-4284, and/or -4326 were increased compared to the control.In some embodiments, the patient was determined to be high risk when theexpression level of miR-30b, miR-32, miR-33a, miR-34a, miR-101,miR-181b, miR-188, miR-191, miR-193b, miR-195, miR-200, miR-200b,miR-362, miR-409, miR-424, miR-425, miR-429, miR-432, miR-592, miR-744,miR-758, miR-1246, miR-3182, miR-3605, miR-3677, miR-4284, and/ormiR-4326 were decreased compared to the control. In some embodiments,the patient was determined to be high risk when the expression level ofCD44v6, AMT, C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1, LYZ, MGAT5, MMP1,MMP9, NOS2, PIGR, PRAC1, RPL39L, RCC1, SPAG16, AMT2, and/or ITGBL1 wereincreased compared to control. In some embodiments, the patient wasdetermined to be high risk when the expression level of CD44v6, AMT,C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1, LYZ, MGAT5, MMP1, MMP9, NOS2, PIGR,PRAC1, RPL39L, RCC1, SPAG16, AMT2, and/or ITGBL1 were decreased comparedto control.

In some embodiments, the method further comprises administering adjuvanttherapy to a patient determined to be high risk.

In some embodiments, the method further comprises measuring theexpression level of the miRNAs in a biological sample from the patient.In some embodiments, the method further comprises comparing theexpression level of the biomarker in the biological sample from thepatient to the expression level of the same biomarker in a controlbiological sample.

In some embodiments, the patient has, is determined to have, or isdiagnosed with stage I, II, III, or IV colorectal cancer. In someembodiments, the patient was determined to have stage I, II, III, or IVcolorectal cancer on the basis of a clinical measurement or biomarkermeasurement described herein. In some embodiments, the patient isdiagnosed with Stage I or II colorectal cancer and does not have lymphnode metastasis. In some embodiments, the patient diagnosed with highrisk is identified as one likely to have or develop distant metastasis,liver metastasis, and/or lymph node metastasis. In some embodiments, thepatient diagnosed with high risk is identified as one likely to developchemoresistance.

In some embodiments, the expression level is normalized. In someembodiments, the biological sample from the patient is a sample from aprimary colorectal cancer tumor. In some embodiments, the biologicalsample from the patient is a blood sample. In some embodiments, thebiological sample from the patient is a serum or plasma sample. In someembodiments, the biological sample from the patient is a biopsy sample.In some embodiments, the biological sample is a biological sampledescribed herein.

In some embodiments, the control is the level of expression or thebiomarker or level of 5hmC DNA modification in a control biologicalsample. In some embodiments, the control biological sample comprisesnormal mucosal tissue. In some embodiments, the control comprises thelevel of expression or the biomarker or level of 5hmC DNA modificationin a non-metastatic colorectal cancer tissue. In some embodiments, thetissue is a primary colorectal cancer tumor. In some embodiments, thecontrol comprises a biological sample from a stage I, II, III, or IVpatient. In some embodiments, the control comprises a level ofexpression or the biomarker or level of 5hmC DNA modification in abiological sample from a patient with non-metastatic or non-progressivecolorectal cancer. In some embodiments, non-progressive colorectalcancer is one that is not classified as having T1, T2 T3, T4, N1, N2,and/or M1. In some embodiments, non-progressive colorectal cancercomprises one that is not characterized by lymph node metastasis. Insome embodiments, non-progressive colorectal cancer comprises one thatis not characterized by distant metastasis. In some embodiments,non-progressive colorectal cancer comprises one that is notcharacterized by lung, liver, breast, or bone metastasis.

In some embodiments, the adjuvant therapy comprises cetuximab,5-fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumumab,afibercept, leucovorin, and/or radiotherapy. In some embodiments, themethod further comprises surgical resection of the primary tumor ormetastatic tumor. In some embodiments, the patient does not have and/orhas not been diagnosed with lymph node metastasis and/or distantmetastsis. In some embodiments, the method further comprises calculatinga risk score based on the expression levels of the miRNAs in thebiological sample from the patient. In some embodiments, the risk scoreis compared to a cut-off value.

In some embodiments, the patient was determined to have differentialexpression of one or more (or all of) miR-409, miR-432, and miR-758. Insome embodiments, the patient was determined to have differentialexpression of miR-758. In some embodiments, the patient was determinedto have differential express of one or more of (or all of) miR-191,miR-200b, miR-30b, miR-33a, miR-362, miR-429, and miR-744. In someembodiments, the patient was determined to have differential expressionof miR-191, miR-200b, miR-33a, miR-429, and miR-744. In someembodiments, the patient was determined to have differential expressionof one or more of (or all of) miR-32, miR-181b, miR-188, miR-193b,miR-195, miR-424, miR-425, miR-592, miR-3677, and/or miR-4326. In someembodiments, the patient was determined to have differential expressionof miR-32, miR-181b, miR-188, miR-193b, miR-195, miR-424, miR-425,miR-592, miR-3677, and miR-4326. In some embodiments, the patient wasdetermined to have one or more risk factors selected from poorlydifferentiated tissues, increased tumor depth; lymphatic invasion, andvenous invasion. In some embodiments, the patient was determined to nothave one or more risk factors selected from poorly differentiatedtissues, increased tumor depth; lymphatic invasion, and venous invasion.In some embodiments, the patient was determined to have differentialexpression of one or more of (or all of) miR-32, miR-181b, miR-188,miR-193b, miR-195, miR-424, miR-425, and miR-592. In some embodiments,the patient was determined to have differential expression of one ormore of miR-1246; miR-34a, miR-101, miR-200, miR-3605, miR-3182, andmiR-4284. In some embodiments, the patient was determined to haveincreased expression of CD44v6 compared to a control. In someembodiments, the patient was determined to have differential expressionof one or more of (or all of) AMT, C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1,LYZ, MGAT5, MMP1, MMP9, NOS2, PIGR, PRAC1, RPL39L, RCC1, and SPAG16. Insome embodiments, the patient was determined to have differentialexpression of one or more of C2CD4A, DEFA6, MGAT5, MMP9, SPAG16, FOXA1,AMT, PRAC1, and RCC1. In some embodiments, the patient was determined tohave differential expression of C2CD4A, DEFA6, MGAT5, MMP9, SPAG16,FOXA1, AMT, PRAC1, and RCC1. In some embodiments, the patient wasdetermined to have differential expression of one or more of (or all of)AMT2, MMP9, DEFA6, FOXA1, MGAT5, C2CD4A, RCC1, LYZ, MMP1, NOS2, PIGR,and CYP2B6. In some embodiments, the patient was determined to havedifferential expression of AMT2, MMP9, DEFA6, FOXA1, MGAT5, C2CD4A,RCC1, LYZ, MMP1, NOS2, PIGR, and CYP2B6. In some embodiments, thepatient was determined to have differential expression of one or more ofAMT2, MMP9, FOXA1, C2CD4A, RCC1, LYZ, MMP1, and PIGR. In someembodiments, the patient was determined to have differential expressionof AMT2, MMP9, FOXA1, C2CD4A, RCC1, LYZ, MMP1, and PIGR. In someembodiments, the patient was determined to have differential expressionof one or more of AMT2, MMP9, FOXA1, RCC1, LYZ, MMP1, and PIGR. In someembodiments, the patient was determined to have differential expressionof AMT2, MMP9, FOXA1, RCC1, LYZ, MMP1, and PIGR. In some embodiments,the patient was determined to have differential expression of one ormore of (or all of) CCAT1 and CCAT2. In some embodiments, the patientwas determined to have increased CEA expression compared to theexpression in a control. In some embodiments, the patient was determinedto have differential expression of one or more of (or all of) DQ595807,DQ570540, and DQ570999. In some embodiments, the patient was determinedto have a low level of 5hmC modified DNA at one or more of (or all of)P2RX4, CRISPLD2, and FKBP4. In some embodiments, the patient wasdetermined to have an increased level of expression of ITGBL1. In someembodiments, the patient was determined to have an increased level ofexpression of DQ596309 and/or DQ570994 (piRACC).

In some embodiments, the method further comprises predicting that thepatient is likely to survive, likely to have disease free survival,and/or likely to have recurrence free survival when the expression levelof the biomarker in the biological sample from the patient is notsignificantly different than the expression level of the biomarker in acontrol.

In some embodiments, the patient was determined to be high risk when oneor more of (or all of) miR-409, miR-432, and miR-758 were determined tobe differentially expressed in the biological sample from the patient.In some embodiments, the patient was determined to be high risk whenmiR-758 was determined to be differentially expressed in the biologicalsample from the patient. In some embodiments, the patient was determinedto be high risk when one or more of (or all of) miR-191, miR-200b,miR-30b, miR-33a, miR-362, miR-429, and/or miR-744 were determined to bedifferentially expressed in the biological sample from the patient. Insome embodiments, the patient was determined to be high risk whenmiR-191, miR-200b, miR-33a, miR-429, and miR-744 were determined to bedifferentially expressed in the biological sample from the patient. Insome embodiments, the patient was determined to be high risk whenmiR-32, miR-181b, miR-188, miR-193b, miR-195, miR-424, miR-425, miR-592,miR-3677, and/or miR-4326 were determined to be differentially expressedin the biological sample from the patient. In some embodiments, thepatient was determined to be high risk when miR-32, miR-181b, miR-188,miR-193b, miR-195, miR-424, miR-425, miR-592, miR-3677, and miR-4326were determined to be differentially expressed in the biological samplefrom the patient. In some embodiments, the patient was determined to behigh risk when the biological sample from the patient demonstrates poortissue differentiation, increased tumor depth; lymphatic invasion,and/or venous invasion in the biological sample from the patient. Insome embodiments, the patient was determined to be high risk when one ormore of (or all of) miR-32, miR-181b, miR-188, miR-193b, miR-195,miR-424, miR-425, and miR-592 were determined to be differentiallyexpressed in the biological sample from the patient. In someembodiments, the patient was determined to be high risk when one or moreof (or all of) miR-1246; miR-34a, miR-101, miR-200, miR-3605, miR-3182,and miR-4284 were determined to be differentially expressed in thebiological sample from the patient. In some embodiments, the patient wasdetermined to be high risk when the expression of CD44v6 was determinedto be increased in the biological sample from the patient. In someembodiments, the patient was determined to be high risk when one or moreof AMT, C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1, LYZ, MGAT5, MMP1, MMP9,NOS2, PIGR, PRAC1, RPL39L, RCC1, and SPAG16 were determined to bedifferentially expressed in the biological sample from the patient. Insome embodiments, the patient was determined to be high risk when one ormore of C2CD4A, DEFA6, MGAT5, MMP9, SPAG16, FOXA1, AMT, PRAC1, and RCC1was determined to be differentially expressed in the biological samplefrom the patient. In some embodiments, the patient was determined to behigh risk when C2CD4A, DEFA6, MGAT5, MMP9, SPAG16, FOXA1, AMT, PRAC1,and RCC1 were determined to be differentially expressed in thebiological sample from the patient. In some embodiments, the patient wasdetermined to be high risk when one or more of AMT2, MMP9, DEFA6, FOXA1,MGAT5, C2CD4A, RCC1, LYZ, MMP1, NOS2, PIGR, and CYP2B6 was determined tobe differentially expressed in the biological sample from the patient.In some embodiments, the patient was determined to be high risk whenAMT2, MMP9, DEFA6, FOXA1, MGAT5, C2CD4A, RCC1, LYZ, MMP1, NOS2, PIGR,and CYP2B6 was determined to be differentially expressed in thebiological sample from the patient. In some embodiments, the patient wasdetermined to have differential expression of one or more of AMT2, MMP9,FOXA1, C2CD4A, RCC1, LYZ, MMP1, and PIGR was determined to bedifferentially expressed in the biological sample from the patient. Insome embodiments, the patient was determined to have differentialexpression of AMT2, MMP9, FOXA1, C2CD4A, RCC1, LYZ, MMP1, and PIGR wasdetermined to be differentially expressed in the biological sample fromthe patient. In some embodiments, the patient was determined to be highrisk when one or more of AMT2, MMP9, FOXA1, RCC1, LYZ, MMP1, and PIGRwas determined to be differentially expressed in the biological samplefrom the patient. In some embodiments, the patient was determined to behigh risk when AMT2, MMP9, FOXA1, RCC1, LYZ, MMP1, and PIGR wasdetermined to be differentially expressed in the biological sample fromthe patient. In some embodiments, the patient was determined to be highrisk when one or more of CCAT1 and CCAT2 was determined to bedifferentially expressed in the biological sample from the patient. Insome embodiments, the patient was determined to be high risk when CCAT1and CCAT2 was determined to be differentially expressed in thebiological sample from the patient. In some embodiments, the methodfurther comprises determining serum CEA expression. In some embodiments,the patient was determined to be high risk when CEA expression isincreased in the biological sample from the patient. In someembodiments, the patient was determined to be high risk when one or moreof DQ595807, DQ570540, and DQ570999 were determined to be differentiallyexpressed in the biological sample from the patient. In someembodiments, the patient was determined to be high risk when DQ595807,DQ570540, and DQ570999 were determined to be differentially expressed inthe biological sample from the patient. In some embodiments, the patientwas determined to be high risk when one or more of (or all of) P2RX4,CRISPLD2, and FKBP4 were determined to have decreased levels of 5hmC inthe biological sample from the patient. In some embodiments, the patientwas determined to be high risk when ITGBL1 was determined to beincreased in the biological sample from the patient. In someembodiments, the patient was determined to be high risk when DQ596309and/or DQ570994 (piRACC) was determined to be differentially expressedin the biological sample from the patient.

Further aspects relate to a method for treating colorectal cancer in apatient, the method comprising administering one or more of miR-30b,miR-32, miR-33a, miR-34a, miR-101, miR-181b, miR-188, miR-191, miR-193b,miR-195, miR-200, miR-200b, miR-362, miR-409, miR-424, miR-425, miR-429,miR-432, miR-592, miR-744, miR-758, miR-1246, miR-3182, miR-3605,miR-3677, miR-4284, miR-4326, CCAT1, CCAT2; piRNA: DQ596309, DQ570994(piRACC), DQ595807 (DQ595807), DQ570540 (DQ570540), DQ570999 (DQ570999);CD44v6, AMT, C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1, LYZ, MGAT5, MMP1,MMP9, NOS2, PIGR, PRAC1, RPL39L, RCC1, SPAG16, AMT2, and/or ITGBL1 orantagonist, agonists, or modifiers thereof.

In some embodiments, the method further comprises determining a riskscore based on the expression levels of the miRNAs in the biologicalsample from the patient. In some embodiments, the risk score is comparedto a cut-off value.

In some embodiments, the adjuvant therapy comprises or the methodfurther comprises administration of one or more of cetuximab,fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman,afibercept, leucovorin, and radiotherapy. In some embodiments, themethod excludes administration of one or more of cetuximab,fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman,afibercept, leucovorin, and radiotherapy. In some embodiments, theadjuvantherapy comprises or the method further comprises administrationof one or more of cetuximab, fluorouracil, oxaliplatin, irinotecan,bevacizumab, panitumuman, afibercept, leucovorin, and radiotherapy. Insome embodiments, the treatment for advanced colorectal cancer excludesadministration of one or more of cetuximab, fluorouracil, oxaliplatin,irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, andradiotherapy. In some embodiments, the adjuvant therapy comprises or themethod further comprises administration of one or more of cetuximab,fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman,afibercept, leucovorin, and radiotherapy. In some embodiments, theadjuvant therapy excludes or the method excludes administration of oneor more of cetuximab, fluorouracil, oxaliplatin, irinotecan,bevacizumab, panitumuman, afibercept, leucovorin, and radiotherapy.

The expression level or activity level from a control sample may be anaverage value, a normalized value, a cut-off value, or an averagenormalized value. The expression level or activity level may be anaverage or mean obtained from a significant proportion of patientsamples. The expression or activity level may also be an average or meanfrom one or more samples from the patient.

In some embodiments, the method further comprises surgical incision ofthe primary tumor. In some embodiments, the elevated level/increasedexpression or reduced level/decreased expression is at least 1, 1.5, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 50,100, 150, 200, 250, 500, or 1000 fold (or any derivable range therein)or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, or 900% different than the control, or any derivablerange therein.

In some embodiments, the biological sample from the patient is a samplefrom a primary colorectal cancer tumor. In some embodiments, thebiological sample is from a tissue or organ as described herein. Instill further embodiments, the method may comprise obtaining a sample ofthe subject or patient. Non-limiting examples of the sample include atissue sample, a whole blood sample, a urine sample, a saliva sample, aserum sample, a plasma sample, or a fecal sample. In particularembodiments, the sample is a rectum sample, a colon sample or a cecumsample.

In some embodiments the subject or patient is one that has previouslybeen treated for colorectal cancer. In some embodiments, the colorectalcancer is recurrent.

The term subject or patient may refer to an animal (for example amammal), including but not limited to humans, non-human primates,rodents, dogs, or pigs. The methods of obtaining provided herein includemethods of biopsy such as fine needle aspiration, core needle biopsy,vacuum assisted biopsy, incisional biopsy, excisional biopsy, punchbiopsy, shave biopsy or skin biopsy.

In certain embodiments the sample is obtained from a biopsy from rectal,cecum, or colon tissue by any of the biopsy methods previouslymentioned. In other embodiments the sample may be obtained from any ofthe tissues provided herein that include but are not limited to gallbladder, skin, heart, lung, breast, pancreas, liver, muscle, kidney,smooth muscle, bladder, intestine, brain, prostate, esophagus, orthyroid tissue.

Alternatively, the sample may include but not be limited to blood,serum, sweat, hair follicle, buccal tissue, tears, menses, urine, feces,or saliva. In particular embodiments, the sample may be a tissue sample,a whole blood sample, a urine sample, a saliva sample, a serum sample, aplasma sample or a fecal sample.

In certain aspects the sample is obtained from cystic fluid or fluidderived from a tumor or neoplasm. In yet other embodiments the cyst,tumor or neoplasm is in the digestive system. In certain aspects of thecurrent methods, any medical professional such as a doctor, nurse ormedical technician may obtain a biological sample for testing. Infurther aspects of the current methods, the patient or subject mayobtain a biological sample for testing without the assistance of amedical professional, such as obtaining a whole blood sample, a urinesample, a fecal sample, a buccal sample, or a saliva sample.

In further embodiments, the sample may be a fresh, frozen or preservedsample or a fine needle aspirate. In particular embodiments, the sampleis a formalin-fixed, paraffin-embedded (FFPE) sample. An acquired samplemay be placed in short term or long term storage by placing in asuitable medium, excipient, solution, or container. In certain casesstorage may require keeping the sample in a refrigerated, or frozenenvironment. The sample may be quickly frozen prior to storage in afrozen environment. In certain instances the frozen sample may becontacted with a suitable cryopreservation medium or compound. Examplesof cryopreservation mediums or compounds include but are not limited to:glycerol, ethylene glycol, sucrose, or glucose.

Some embodiments further involve isolating nucleic acids such asribonucleic or RNA from a biological sample or in a sample of thepatient. Other steps may or may not include amplifying a nucleic acid ina sample and/or hybridizing one or more probes to an amplified ornon-amplified nucleic acid. The methods may further comprise assayingnucleic acids in a sample. Further embodiments include isolating oranalyzing protein expression in a biological sample for the expressionof the biomarker.

In certain embodiments, a microarray may be used to measure or assay thelevel of the biomarkers in a sample. The methods may further compriserecording the biomarker expression or activity level in a tangiblemedium or reporting the expression or activity level to the patient, ahealth care payer, a physician, an insurance agent, or an electronicsystem.

In some embodiments, methods will involve determining or calculating aprognosis score based on data concerning the expression or activitylevel of one or more of the biomarkers, meaning that the expression oractivity level of one or more of the biomarkers is at least one of thefactors on which the score is based. A prognosis score will provideinformation about the patient, such as the general probability whetherthe patient is sensitive to a particular therapy or has poor survival orhigh chances of recurrence. In certain embodiments, a prognosis value isexpressed as a numerical integer or number that represents a probabilityof 0% likelihood to 100% likelihood that a patient has a chance of poorsurvival or cancer recurrence or poor response to a particulartreatment.

In some embodiments, the prognosis score is expressed as a number thatrepresents a probability of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%likelihood (or any range derivable therein) that a patient has a chanceof poor survival or cancer recurrence or poor response to a particulartreatment. Alternatively, the probability may be expressed generally inpercentiles, quartiles, or deciles.

A difference between or among weighted coefficients or expression oractivity levels or between or among the weighted comparisons may be, beat least or be at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5,11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5,17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380,385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475,480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590,600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710,720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825,830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940,950, 960, 970, 975, 980, 990, 1000 times or -fold (or any rangederivable therein).

In some embodiments, determination of calculation of a diagnostic,prognostic, or risk score is performed by applying classificationalgorithms based on the expression values of biomarkers withdifferential expression p values of about, between about, or at mostabout 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013,0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.020, 0.021, 0.022, 0.023,0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033,0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042, 0.043,0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0.053,0.054, 0.055, 0.056, 0.057, 0.058, 0.059, 0.060, 0.061, 0.062, 0.063,0.064, 0.065, 0.066, 0.067, 0.068, 0.069, 0.070, 0.071, 0.072, 0.073,0.074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.080, 0.081, 0.082, 0.083,0.084, 0.085, 0.086, 0.087, 0.088, 0.089, 0.090, 0.091, 0.092, 0.093,0.094, 0.095, 0.096, 0.097, 0.098, 0.099, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9 or higher (or any range derivable therein). In certainembodiments, the prognosis score is calculated using one or morestatistically significantly differentially expressed biomarkers (eitherindividually or as difference pairs), including expression or activitylevels in a biomarker, gene, or protein.

Further aspects relate to a kit comprising nucleic acid probes fordetecting the expression level of differentially expressed biomarkers ina biological sample; wherein the differentially expressed biomarkerscomprise one or more of: miRNAs: miR-30b, miR-32, miR-33a, miR-34a,miR-101, miR-181b, miR-188, miR-191, miR-193b, miR-195, miR-200,miR-200b, miR-362, miR-409, miR-424, miR-425, miR-429, miR-432, miR-592,miR-744, miR-758, miR-1246, miR-3182, miR-3605, miR-3677, miR-4284,and/or miR-4326; lncRNA: CCAT1 and/or CCAT2; piRNA: DQ596309, DQ570994(piRACC), DQ595807, DQ570540, and/or DQ570999; 5hmC DNA modificationlevels of genes: P2RX4, CRISPLD2, and/or FKBP4; and mRNA gene expressionand/or protein level of: CD44v6, AMT, C2CD4A, CYP2B6, DEFA6, FOXA1,GSTT1, LYZ, MGAT5, MMP1, MMP9, NOS2, PIGR, PRAC1, RPL39L, RCC1, SPAG16,AMT2, and/or ITGBL1.

In some embodiments, the differentially expressed biomarkers consist ofmiR-409, miR-432, and miR-758. In some embodiments, the differentiallyexpressed biomarkers consist of miR-758. In some embodiments, thedifferentially expressed biomarkers consist of miR-191, miR-200b,miR-30b, miR-33a, miR-362, miR-429, and miR-744. In some embodiments,the differentially expressed biomarkers consist of miR-191, miR-200b,miR-33a, miR-429, and miR-744. In some embodiments, the differentiallyexpressed biomarkers consist of miR-32, miR-181b, miR-188, miR-193b,miR-195, miR-424, miR-425, miR-592, miR-3677, and miR-4326. In someembodiments, the differentially expressed biomarkers consist of miR-32,miR-181b, miR-188, miR-193b, miR-195, miR-424, miR-425, and miR-592. Insome embodiments, the differentially expressed biomarkers consist ofmiR-1246; miR-34a, miR-101, miR-200, miR-3605, miR-3182, and miR-4284.In some embodiments, the differentially expressed biomarker consists ofCD44v6. In some embodiments, the differentially expressed biomarkersconsist of AMT, C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1, LYZ, MGAT5, MMP1,MMP9, NOS2, PIGR, PRAC1, RPL39L, RCC1, and SPAG16. In some embodiments,the differentially expressed biomarkers consist of C2CD4A, DEFA6, MGAT5,MMP9, SPAG16, FOXA1, AMT, PRAC1, and RCC1. In some embodiments, thedifferentially expressed biomarkers consist of AMT2, MMP9, DEFA6, FOXA1,MGAT5, C2CD4A, RCC1, LYZ, MMP1, NOS2, PIGR, and CYP2B6. In someembodiments, the differentially expressed biomarkers consist of AMT2,MMP9, FOXA1, C2CD4A, RCC1, LYZ, MMP1, and PIGR. In some embodiments, thedifferentially expressed biomarkers consist of AMT2, MMP9, FOXA1, RCC1,LYZ, MMP1, and PIGR. In some embodiments, the differentially expressedbiomarkers consist of CCAT1 and CCAT2. In some embodiments, thedifferentially expressed biomarkers consist of DQ595807, DQ570540, andDQ570999. In some embodiments, the differentially expressed biomarkersconsist of P2RX4, CRISPLD2, and FKBP4. In some embodiments, thedifferentially expressed biomarker consists of ITGBL1. In someembodiments, the differentially expressed biomarkers consist of DQ596309and DQ570994 (piRACC).

In some embodiments, the probes are labeled. In some embodiments, thekit further comprises nucleic acid probes for detecting a control. Insome embodiments, the control comprises a RNA, miRNA, or protein notdifferentially expressed in colorectal cancer. In some embodiments, theprobe comprises nucleic acid primers that are capable of amplifying theRNA or a cDNA made from the RNA by PCR. In some embodiments, the kitfurther comprises reagents for performing one or more of reversetranscriptase PCR, DNA amplification by PCR, and real-time PCR. In someembodiments, the kit further comprises instructions for use.

Any of the methods described herein may be implemented on tangiblecomputer-readable medium comprising computer-readable code that, whenexecuted by a computer, causes the computer to perform one or moreoperations. In some embodiments, there is a tangible computer-readablemedium comprising computer-readable code that, when executed by acomputer, causes the computer to perform operations comprising: a)receiving information corresponding to an expression or activity levelof a gene, biomarker or protein in a sample from a patient; and b)determining a difference value in the expression or activity levelsusing the information corresponding to the expression or activity levelsin the sample compared to a control or reference expression or activitylevel for the gene.

In other aspects, tangible computer-readable medium further comprisecomputer-readable code that, when executed by a computer, causes thecomputer to perform one or more additional operations comprising makingrecommendations comprising: wherein the patient in the step a) is underor after a first treatment for colorectal cancer, administering the sametreatment as the first treatment to the patient if the patient does nothave increased expression or activity level; administering a differenttreatment from the first treatment to the patient if the patient hasincreased expression or activity level.

In some embodiments, receiving information comprises receiving from atangible data storage device information corresponding to the expressionor activity levels from a tangible storage device. In additionalembodiments the medium further comprises computer-readable code that,when executed by a computer, causes the computer to perform one or moreadditional operations comprising: sending information corresponding tothe difference value to a tangible data storage device, calculating aprognosis score for the patient, treating the patient with a traditionalcolorectal therapy if the patient does not have expression or activitylevels, and/or or treating the patient with an alternative colorectaltherapy if the patient has increased expression or activity levels.

The tangible, computer-readable medium further comprisecomputer-readable code that, when executed by a computer, causes thecomputer to perform one or more additional operations comprisingcalculating a prognosis score for the patient. The operations mayfurther comprise making recommendations comprising: administering atreatment comprising a thymidylate synthase inhibitor to a patient thatis determined to have a decreased expression or activity level.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A-D: Schematic design of miRNA discovery (a-c); Log 2 expressionlevel of differentially expressed miRNAs in recurrent and non-recurrentcolorectal cancer tissues. These results are further discussed inExample 1.

FIG. 2A-C: Relative expression of differentially expressed miRNAs inrecurrent and non-recurrent colorectal cancer tissues (a). Correlationof disease free survival (DFS) and overall survival (OS) with indicatedmiRNA expression (b). These results are further discussed in Example 1.

FIG. 3: ROC analysis, correlation with disease free survival, andpre-operative and post-operative serum levels of miR-758. These resultsare further discussed in Example 1.

FIG. 4: Correlation of disease free survival and expression levels ofvarious miRNAs. These results are further discussed in Example 1.

FIG. 5: Schematic of experimental design for discovery of colorectalcancer microRNA markers for prognosis—discovery from TCGA. These resultsare further discussed in Example 2.

FIG. 6: Clinical validation in NCCH cohort for Stage 2 and 3 colorectalcancer tissues. These results are further discussed in Example 2.

FIG. 7: Clinical validation for Stage 2 and 3 colorectal cancer tissues.These results are further discussed in Example 2.

FIG. 8A-C: Analysis of miRNA expression for correlation with colorectalcancer. Shown are results of expression analysis (a), the sensitivityand specificity ROC analysis for the prediction of lymph node metastasis(LNM) of each miRNA alone (b), of the combination of mir-32, mir-181b(-1 and -2), mir-188, mir-193b, mir-195, mir-424, mir-425, mir-592,mir-3677, and mir-4326 in T1-2 CRC (10-miRNA signature; c; left panel),of the combination of mir-32, mir-181b (-1 and -2), mir-188, mir-193b,mir-195, mir-424, mir-425, mir-592, mir-3677, and mir-4326 in T1 CRCpatients of the TCGA database (c; middle panel), and of the combinationof mir-32, mir-181b (-1 and -2), mir-188, mir-193b, mir-195, mir-424,mir-425, and mir-592 in tissues of primary and LNM tissues (c; rightpanel). These results are further discussed in Example 3.

FIG. 9A-C: Analysis of miRNA expression for correlation with colorectalcancer. Shown are ROC analyses of the sensitivity/specificity of the10-gene miRNA signature in predicting LNM in samples from the KumamotoUniv. cohort (a; AUC=0.908) and Univ. Tokyo cohort (b; AUC=0.842). ThemiRNA signature was better at predicting LMN than conventional riskfactors such as tumor depth, lymphatic invasion, venous invasion, andpoorly histology (c; top left) or other clinical measurements such asserum CEA, CT, or tumor budding (c; top right). When combined with theconventional risk factors (tumor depth, lymphatic invasion, venousinvasion, and poorly histology) using logistic regression model, thismodel showed better AUC value to predict LNM in T1 CRC (c; bottom leftand right). These results are further discussed in Example 3.

FIG. 10: Schematic diagram of miRNA biomarker discovery and analysis.These results are further discussed in Example 3.

FIG. 11A-B: Kumamoto Univ. cohort. (a) Expression levels of variousmiRNA between LNM positive and negative samples and (b) sensitivity andspecificity ROC analysis for the prediction of lymph node metastasis(LNM) of each miRNA alone from the of the Kumamoto Univ. cohort. Theseresults are further discussed in Example 3.

FIG. 12A-B: Univ. Tokyo cohort. (a) Expression levels of various miRNAbetween LNM positive and negative samples and (b) sensitivity andspecificity ROC analysis for the prediction of lymph node metastasis(LNM) of each miRNA alone from the of the Univ. Tokyo cohort. Theseresults are further discussed in Example 3.

FIG. 13: Relative expression of miR-1246 in CD44v6⁺ colorectal cancerstem cells: HT29 (left), HCT116 (middle), and 5-FU resistant (right).These results are further discussed in Example 4.

FIG. 14: The expression of miR-1246 was significantly elevated in CRCtissues compared to corresponding normal mucosa (left and middle), andthis occurred in a stage-dependent manner in primary CRCs (right). Theseresults are further discussed in Example 4.

FIG. 15A-C: High miR-1246 expression resulted in poor overall (a) andpoor disease free survival (b). The expression of CD44v6 positivelycorrelated with miR-1246 in CRC tissues (c). These results are furtherdiscussed in Example 4.

FIG. 16: Tokyo clinical cohort 1: ROC analysis. 19 LN positive and 117LN negative samples were in the cohort. The analysis yielded an AUCvalue of 0.9. This analysis is further described in Example 5.

FIG. 17: Tokyo clinical cohort 1: Boxplots of mRNA normalized expressionin lymph node negative (LN−) and lymph node positive (LN+) samples. Thisanalysis is further described in Example 5.

FIG. 18A-B: Tokyo clinical cohort 1: ROC curves for individual mRNAs.This analysis is further described in Example 5.

FIG. 19: Kumamoto clinical cohort 2: ROC analysis. 8 LN positive and 59LN negative samples were in the cohort. The analysis yielded an AUCvalue of 0.896. This analysis is further described in Example 5.

FIG. 20: Kumamoto clinical cohort 2: Boxplots of mRNA normalizedexpression in lymph node negative (LN−) and lymph node positive (LN+)samples. This analysis is further described in Example 5.

FIG. 21A-B: Kumamoto clinical cohort 2: ROC curves for individual mRNAs.This analysis is further described in Example 5.

FIG. 22: TMDU clinical cohort 3: ROC analysis. 4 LN positive and 35 LNnegative samples were in the cohort. The analysis yielded an AUC valueof 1.0. This analysis is further described in Example 5.

FIG. 23: TMDU clinical cohort 3: Boxplots of mRNA normalized expressionin lymph node negative (LN−) and lymph node positive (LN+) samples. Thisanalysis is further described in Example 5.

FIG. 24A-B: TMDU clinical cohort 3: ROC curves for individual mRNAs.This analysis is further described in Example 5.

FIG. 25A-B: Validation on TCGA; ROC curves for cohort with (a) T1 CRCpatients (2 LN+, 8 LN−); AUC=1.0 (ROC), 1.0 (Precision) and (b) T1 andT2 CRC patients (16 LN+, 109 LN−); AUC=0.95 (ROC), 0.85 (precision)using 16 gene classifier described in Example 5.

FIG. 26A-B: Boxplots of expression of indicated mRNA of LM− (left box ofeach graph) and LM+ (right box of each graph) samples of the 16 genes inthe 16 gene classifier described in Example 5.

FIG. 27A-B: ROC analysis of each mRNA alone of the 16-gene classifierdescribed in Example 5.

FIG. 28A-B: Validation on CIT/GSE39582: ROC curves for cohort with (a)T1&T2 CRC patients (2 LN+, 9 LN−); AUC=1.0 (ROC), 1.0 (Precision) and(b) T1 and T2 CRC patients (18 LN+, 38 LN−); AUC=0.93 (ROC), 0.85(precision) using 15 gene classifier described in Example 5.

FIG. 29A-C: Boxplots of expression of indicated mRNA of LM− (left box ofeach graph) and LM+ (right box of each graph) samples of the 15 genes inthe 15 gene classifier described in Example 5.

FIG. 30A-C: ROC analysis of each mRNA alone of the 15-gene classifierdescribed in Example 5.

FIG. 31: Use Recursive Feature Elimination based on 5-foldcross-validation of Random Forest algorithm, 9 were kept as a 9-geneclassifier. These results are further discussed in Example 5.

FIG. 32A-B: Validation on TCGA. ROC curves for cohort with (a) TCGAcohort with T1 CRC patients (2 LN+, 18 LN−); AUC=1.0 (ROC), 1.0(Precision); (b) TCGA cohort with T1 and T2 CRC patients (16 LN+, 109LN−); AUC=0.94 (ROC), 0.78 (precision); (c) CIT/GSE39582 cohort with T1CRC patients (2 LN+, 9 LN−); AUC=1.0 (ROC), 1.0 (Precision); and (d)CIT/GSE39582 cohort with T1 and T2 CRC patients (18 LN+, 38 LN−);AUC=0.86 (ROC), 0.74 (precision) using 9 gene classifier described inExample 5.

FIG. 33A-D: The testing and validation phase of this study. a) CCAT1expression and association with recurrence free survival (RFS) andoverall survival (OS) in cohort 2. High CCAT1 expression was associatedwith poor RFS and poor OS (P=0.049 and 0.028, respectively). b) CCAT2expression and association with RFS and OS in cohort 2. High CCAT2expression was associated with poor RFS and poor OS (P=0.022 and 0.015,respectively). c) CCAT1 expression and association with RFS and OS incohort 3. High CCAT1 expression was significantly association with RFSand OS (P<0.001 and 0.011, respectively). d) CCAT2 expression andassociation with RFS and OS in cohort 3. High CCAT2 expression wassignificantly association with RFS and OS (P=0.010 and 0.025,respectively). These results are further discussed in Example 6.

FIG. 34A-C: Combination of CCAT1, CCAT2 expression and association withRFS, OS. a) Survival curves plotting co-expression of CCAT1 and CCAT2lncRNAs versus recurrence free survival (RFS) and overall survival (OS)in 135 patients with colorectal cancer. Patients whose tumors expressedhigh levels of both CCAT1 and CCAT2 had poorer RFS compared with thosewho express high levels of either CCAT1 or CCAT2 (P=0.049) and those whoexpress low levels of CCAT1 and CCAT2 lncRNAs (P<0.001). OS showed thesame trends, with patients expressing low levels of CCAT1 and CCAT2having a better OS than those expression high levels of CCAT1 or CCAT2(P=0.038) and those with high levels of expression of both lncRNAs(P=0.002). b) Receiver operating characteristic analysis comparing theaccuracy of predicting recurrence in 5 years for patients with stageI-III CRC. Expression of CCAT1, CCAT2, and several clinicopathologicalfactors, and combination model of CCAT1, CCAT2 and CEA expression wereinvestigated. Combination model showed the highest area under the curve(AUC) of 0.793. c) The association of RFS with combination model ofCCAT1, CCAT2, and CEA expression in tumor tissues from stage II andstage III colorectal cancer patients. High levels of combination modelshowed poorer RFS than those with low expression in both stage II andstage III patients (P=0.034 and 0.001, respectively). These results arefurther discussed in Example 6.

FIG. 35A-B: The study design, and the screening phase of the study. a)Study design. b) The long non-coding RNAs (lncRNAs) located in the8q24.21 locus. These results are further discussed in Example 6.

FIG. 36A-B: The screening phase of the study. Expression of twelvelncRNAs was compared between 20 CRC tissues and their matched adjacentnormal mucosa. CCAT1, CCAT1-L, CCAT2, PVT1, and CASC19 lncRNAs weresignificantly overexpressed in cancer tissues compared to the matchedcontrols (P=0.039, <0.001, 0.018, <0.001, 0.002, respectively). Theseresults are further discussed in Example 6.

FIG. 37A-C: The testing phase of the study. CCAT1-L, PVT1, and CASC19lncRNA expressions were evaluated in 125 colorectal cancer tissues, andtheir associations with recurrence free survival (RFS) and overallsurvival (OS) were compared using Kaplan-Meier curve. a) High levels ofCCAT1-L were significantly associated with poor RFS (P=0.048), althoughnot with OS (P=0.352). b) c) PVT1 and CASC19 showed no significantassociation with RFS (P=0.178 and 0.087, respectively) or OS (P=0.113and 0.290, respectively). These results are further discussed in Example6.

FIG. 38: The association between MYC expression and CCAT1 and CCAT2expressions. Correlation between MYC expression and expression of CCAT1and CCAT2 lncRNA levels. Expression of both CCAT1 and CCAT2 were highlycorrelated with MYC expression (r=0.66; P<0.001, r=0.74; P<0.001,Pearson's correlation). These results are further discussed in Example6.

FIG. 39 depicts the 5mC demethylation pathway. These results are furtherdiscussed in Example 8.

FIG. 40A-E shows the log fold change of 5hmC in each of the indicatedgenes in the paired cohort (25 samples). Shown in each graph is the foldchange in normal (left) and cancer (right) samples. These results arefurther discussed in Example 8.

FIG. 41 shows the global 5hmC level in tumor (c) and normal (n) samples(left) and the stage specific global 5hmC levels (right) of independentcohort 1. 100 colorectal cancer samples and 48 normal samples were incohort 1. These results are further discussed in Example 8.

FIG. 42 shows the 5hmC levels of the indicated genes in each stage ofsamples from paired cohort. These results are further discussed inExample 8.

FIG. 43A-C shows the ability of the indicated gene biomarkers to bepredictive of overall survival in colorectal cancer (paired cohort).These results are further discussed in Example 8.

FIG. 44A-C shows the ability of the indicated gene biomarkers to bepredictive of disease free survival in colorectal cancer (pairedcohort). These results are further discussed in Example 8.

FIG. 45: The association of the indicated gene biomarkers metastasis incolorectal cancer paired cohort). These results are further discussed inExample 8.

FIG. 46: Global 5hmC level in tumor (c) and normal (n) samples (left)and the stage specific global 5hmC levels (right) of the cohort 2 (152samples; Normal=48). These results are further discussed in Example 8.

FIG. 47 shows the 5hmC levels of the indicated genes in each CRC stageof samples from the largercohort 2. These results are further discussedin Example 8.

FIG. 48A-C shows the ability of the indicated gene biomarkers to bepredictive of overall survival in colorectal cancer Larger cohort 2.These results are further discussed in Example 8.

FIG. 49A-C shows the ability of the indicated gene biomarkers to bepredictive of disease free survival in colorectal cancer (cohort 2).These results are further discussed in Example 8.

FIG. 50: TCGA data RNASeq expression. TCGA dataset was used to examinethe expression pattern of these genes. 2 out of 3 genes (P2RX4 andCRISPLD2) had reduced expression in CRC tissues compared to normaltissue, which corroborated with the finding of reduced 5hmC levels inthese tissues. These results are further discussed in Example 8.

FIG. 51A-B: Identification of cancer-related piRNAs in CRC. (A) Theexpression of candidate piRNAs were validated in a subset of 20 cancerand paired NM specimens from Mie cohort. (B) The expression of DQ570994and DQ596309 were further confirmed in Shanghai (cohort I) and Okayamacohort (cohort II). DQ570994 were consistently higher in cancer versusnormal tissues in each cohort, and we named this piRNA as piRNA DQ570994associated with colorectal cancer (piRACC). **P<0.01, Wilcoxon pairedtest. These results are further discussed in Example 10.

FIG. 52A-D: piRACC exhibits pan-cancer pattern expression and correlateswith poor prognosis in CRC patients. (A-B) TCGA datasets showed piRACCis significantly upregulated in different cancer types. **P<0.01,Wilcoxon paired test. The prognostic significance of piRACC wasevaluated in colorectal cancer patients from TCGA datasets (C) andclinical testing and validation cohorts (D). ROC curve analysis yieldedoptimal cutoff expression values to discriminate dead or alive patients.Colorectal cancer patients were thereafter divided into high- and lowexpression groups based upon these cut off values. The OS analysis wasperformed by Kaplan-Meier test and the log-rank method (**P<0.05, HR:Hazard Ratio). These results are further discussed in Example 10.

FIG. 53A-E: piRACC promotes cell growth, colony formation, migration andinvasion and inhibits apoptosis in vitro. HCT116 and SW480 cells weretransfected with either piRACC RNA oligos, antisense or scrambledcontrol. The treated cells or control cells were subsequently used forMTT assay (A), colony formation assay (B), Ki-67 staining (C), Migrationand invasion assay (D) and apoptosis assay (E). All the experiments wereperformed biological triplicate. (*P<0.05, **P<0.01; independent t-testwas used to compare control and treated cells). These results arefurther discussed in Example 10.

FIG. 54A-C: KEGG, Gene Ontology (GO) and Ingenuity Pathway Analysis(IPA) for the differentially expressed genes between piRACC inhibitionHCT116 cells and control cells. (A) KEGG analysis for the up-regulatedgenes (B) Go annotation of up-regulated genes with top 10 enrichmentpathways covering domains of biological processes, cellular componentsand molecular functions. (C) IPA analysis for the upregulated genes tointerrogate the function of piRACC in CRC. These results are furtherdiscussed in Example 10.

FIG. 55A-B: Identification of piRACC target mRNAs. (A) miRANDA and RNA22was used to predict the binding of piRACC to potential targets. (B) qPCRwas performed to confirm the expression change of target genes afterpiRACC overexpression or knockdown in HCT116 and SW480 cells. (n=3,*P<0.05, **P<0.01, independent t-test was used to compare control andtreated cells). These results are further discussed in Example 10.

FIG. 56A-B: The correlation between piRACC and its target genes in CRCtissues. qPCR was performed to evaluate the expression correlationbetween piRACC and its targets in CRC tissues. (n=159, *P<0.05,**P<0.01; Spearman's rank correlation (ρ) was used for the correlationanalysis). These results are further discussed in Example 10.

FIG. 57: PIWIL1 and PIWIL4 are overexpressed in CRC. The representativeIHC staining of PIWIL1 and PIWIL4 in CRC and normal tissues (provided byProtein atlas database). These results are further discussed in Example10.

FIG. 58: Gene Ontology (GO) analysis for the down-regulated genes. Goannotation of top 10 enrichment pathways covering domains of biologicalprocesses, cellular components and molecular functions. These resultsare further discussed in Example 10.

FIG. 59A-C: Prediction of piRACC's target by miRanda. The representativeimages showed the binding sites between piRACC and its targets. Theseresults are further discussed in Example 10.

FIG. 60A-B: Prediction of piRACC's target by RNA22. The representativeimages showed the binding sites between piRACC and its targets. Theseresults are further discussed in Example 10.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Dysregulated expression of microRNAs (miRNAs) has emerged as a hallmarkfeature in human cancers. Aspects of the disclosure relate to methodsfor selecting optimal therapy for a patient from several alternativetreatment options. A major clinical challenge in cancer treatment is toidentify the subset of patients who will benefit from a therapeuticregimen, both in metastatic and adjuvant settings. The number ofanti-cancer drugs and multi-drug combinations has increasedsubstantially in the past decade, however, treatments continue to beapplied empirically using a trial-and-error approach. Here methods andcompositions are provided to determine the optimal treatment option forcancer patients.

I. DEFINITIONS

As used herein, the term “antibody” encompasses antibodies and antibodyfragments thereof, derived from any antibody-producing mammal (e.g.,mouse, rat, rabbit, and primate including human), that specifically bindto an antigenic polypeptide. Exemplary antibodies include polyclonal,monoclonal and recombinant antibodies; multispecific antibodies (e.g.,bispecific antibodies); humanized antibodies; murine antibodies;chimeric, mouse-human, mouse-primate, primate-human monoclonalantibodies; and anti-idiotype antibodies, and may be any intact moleculeor fragment thereof.

“Prognosis” refers to as a prediction of how a patient will progress,and whether there is a chance of recovery. “Cancer prognosis” generallyrefers to a forecast or prediction of the probable course or outcome ofthe cancer, with or without a treatment. As used herein, cancerprognosis includes the forecast or prediction of any one or more of thefollowing: duration of survival of a patient susceptible to or diagnosedwith a cancer, duration of recurrence-free survival, duration ofprogression free survival of a patient susceptible to or diagnosed witha cancer, response rate in a group of patients susceptible to ordiagnosed with a cancer, duration of response in a patient or a group ofpatients susceptible to or diagnosed with a cancer, and/or likelihood ofmetastasis in a patient susceptible to or diagnosed with a cancer.Prognosis also includes prediction of favorable responses to cancertreatments, such as a conventional cancer therapy. A response may beeither a therapeutic response (sensitivity or recurrence-free survival)or a lack of therapeutic response (residual disease, which may indicateresistance or recurrence).

The term substantially the same or not significantly different refers toa level of expression that is not significantly different than what itis compared to. Alternatively, or in conjunction, the term substantiallythe same refers to a level of expression that is less than 2, 1.5, or1.25 fold different than the expression or activity level it is comparedto.

By “subject” or “patient” is meant any single subject for which therapyis desired, including humans, cattle, dogs, guinea pigs, rabbits,chickens, and so on. Also intended to be included as a subject are anysubjects involved in clinical research trials not showing any clinicalsign of disease, or subjects involved in epidemiological studies, orsubjects used as controls.

The term “disease free survival” is a clinical endpoint and is usuallyused to analyze the results of the treatment for the localized diseasewhich renders the patient apparently disease free, such as surgery orsurgery plus adjuvant therapy. In the disease-free survival, the eventis relapse rather than death. The people who relapse are still survivingbut they are no longer disease-free. Just as in the survival curves notall patients die, in “disease-free survival curves” not all patientsrelapse and the curve may have a final plateau representing the patientswho didn't relapse after the study's maximum follow-up. Because thepatients survive for at least some time after the relapse, the curve forthe actual survival would look better than disease free survival curve.

The term “primer” or “probe” as used herein, is meant to encompass anynucleic acid that is capable of priming the synthesis of a nascentnucleic acid in a template-dependent process. Typically, primers areoligonucleotides from ten to twenty and/or thirty base pairs in length,but longer sequences can be employed. Primers may be provided indouble-stranded and/or single-stranded form, although thesingle-stranded form is preferred.

As used herein, “increased expression” or “elevated expression” or“decreased expression” refers to an expression level of a biomarker inthe subject's sample as compared to a reference level representing thesame biomarker or a different biomarker. In certain aspects, thereference level may be a reference level of expression from anon-cancerous tissue from the same subject. Alternatively, the referencelevel may be a reference level of expression from a different subject orgroup of subjects. For example, the reference level of expression may bean expression level obtained from a sample (e.g., a tissue, fluid orcell sample) of a subject or group of subjects without cancer, or anexpression level obtained from a non-cancerous tissue of a subject orgroup of subjects with cancer. The reference level may be a single valueor may be a range of values. The reference level of expression can bedetermined using any method known to those of ordinary skill in the art.In some embodiments, the reference level is an average level ofexpression determined from a cohort of subjects with cancer or withoutcancer. The reference level may also be depicted graphically as an areaon a graph. In certain embodiments, a reference level is a normalizedlevel.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. In some embodimentsit is contemplated that an numerical value discussed herein may be usedwith the term “about” or “approximately.”

II. COLORECTAL CANCER STAGING AND TREATMENTS

Methods and compositions may be provided for treating colorectal cancerwith particular applications of biomarker expression or activity levels.Based on a profile of biomarker expression or activity levels, differenttreatments may be prescribed or recommended for different cancerpatients.

A. Cancer Staging

Colorectal cancer, also known as colon cancer, rectal cancer, or bowelcancer, is a cancer from uncontrolled cell growth in the colon or rectum(parts of the large intestine), or in the appendix. Certain aspects ofthe methods are provided for patients that are stage I-IV colorectalcancer patients. In particular aspects, the patient is a stage II or IIIpatient. In a further embodiment, the patient is a stage I or IIpatient. In a further embodiment, the patient is a stage I, II, or IIIpatient.

The most common staging system is the TNM (for tumors/nodes/metastases)system, from the American Joint Committee on Cancer (AJCC). The TNMsystem assigns a number based on three categories. “T” denotes thedegree of invasion of the intestinal wall, “N” the degree of lymphaticnode involvement, and “M” the degree of metastasis. The broader stage ofa cancer is usually quoted as a number I, II, III, IV derived from theTNM value grouped by prognosis; a higher number indicates a moreadvanced cancer and likely a worse outcome. Details of this system arein the graph below:

AJCC TNM TNM stage criteria for colorectal stage stage cancer Stage 0Tis N0 M0 Tis: Tumor confined to mucosa; cancer-in-situ Stage I T1 N0 M0T1: Tumor invades submucosa Stage I T2 N0 M0 T2: Tumor invadesmuscularis propria Stage II-A T3 N0 M0 T3: Tumor invades subserosa orbeyond (without other organs involved) Stage II-B T4 N0 M0 T4: Tumorinvades adjacent organs or perforates the visceral peritoneum StageIII-A T1-2 N1 M0 N1: Metastasis to 1 to 3 regional lymph nodes. T1 orT2. Stage III-B T3-4 N1 M0 N1: Metastasis to 1 to 3 regional lymphnodes. T3 or T4. Stage III-C any T, N2 M0 N2: Metastasis to 4 or moreregional lymph nodes. Any T. Stage IV any T, any N, M1: Distantmetastases present. Any T, M1 any N.

B. Therapy

For people with localized and/or early colorectal cancer, the preferredtreatment is complete surgical removal with adequate margins, with theattempt of achieving a cure. This can either be done by an openlaparotomy or sometimes laparoscopically. Sometimes chemotherapy is usedbefore surgery to shrink the cancer before attempting to remove it(neoadjuvant therapy). The two most common sites of recurrence ofcolorectal cancer is in the liver and lungs. In some embodiments, thetreatment of early colorectal cancer excludes chemotherapy. In furtherembodiments, the treatment of early colorectal cancer includesneoadjuvant therapy (chemotherapy or radiotherapy before the surgicalremoval of the primary tumor), but excludes adjuvant therapy(chemotherapy and/or radiotherapy after surgical removal of the primarytumor.

In both cancer of the colon and rectum, chemotherapy may be used inaddition to surgery in certain cases. In rectal cancer, chemotherapy maybe used in the neoadjuvant setting.

In certain embodiments, there may be a decision regarding thetherapeutic treatment based on biomarker expression. Chemotherapy basedon antimetabolites or thymidylate synthase inhibitors such asfluorouracil (5-FU) have been the main treatment for metastaticcolorectal cancer. Major progress has been made by the introduction ofregimens containing new cytotoxic drugs, such as irinotecan oroxaliplatin. The combinations commonly used, e.g., irinotecan,fluorouracil, and Jeucovorin (FOLFIRI) and oxaliplatin, fluorouracil,and leucovorin (FOLFOX) can reach an objective response rate of about50%. However, these new combinations remain inactive in one half of thepatients and, in addition, resistance to treatment appear in almost allpatients who were initially responders. More recently, two monoclonalantibodies targeting vascular endothelial growth factor Avastin®(bevacizumab) (Genentech Inc., South San Francisco Calif.) and epidermalgrowth factor receptor Erbitux® (cetuximab) (Imclone Inc. New York City)have been approved for treatment of metastatic colorectal cancer but arealways used in combination with standard chemotherapy regimens. In someembodiments, the cancer therapy may include one or more of the chemicaltherapeutic agents including thymidylate synthase inhibitors orantimetabolites such as fluorouracil (5-FU), alone or in combinationwith other therapeutic agents.

For example, in some embodiments, the first treatment to be tested forresponse therapy may be antimetabolites or thymidylate synthaseinhibitors, prodrugs, or salts thereof. In some embodiments, thistreatment regimen is for advanced cancer. In some embodiments, thistreatment regimen is excluded for early cancer.

Antimetabolites can be used in cancer treatment, as they interfere withDNA production and therefore cell division and the growth of tumors.Because cancer cells spend more time dividing than other cells,inhibiting cell division harms tumor cells more than other cells.Anti-metabolites masquerade as a purine (azathioprine, mercaptopurine)or a pyrimidine, chemicals that become the building-blocks of DNA. Theyprevent these substances becoming incorporated in to DNA during the Sphase (of the cell cycle), stopping normal development and division.They also affect RNA synthesis. However, because thymidine is used inDNA but not in RNA (where uracil is used instead), inhibition ofthymidine synthesis via thymidylate synthase selectively inhibits DNAsynthesis over RNA synthesis. Due to their efficiency, these drugs arethe most widely used cytostatics. In the ATC system, they are classifiedunder L01B. In some embodiments, this treatment regimen is for advancedcancer. In some embodiments, this treatment regimen is excluded forearly cancer.

Thymidylate synthase inhibitors are chemical agents which inhibit theenzyme thymidylate synthase and have potential as an anticancerchemotherapy. As an anti-cancer chemotherapy target, thymidylatesynthetase can be inhibited by the thymidylate synthase inhibitors suchas fluorinated pyrimidine fluorouracil, or certain folate analogues, themost notable one being raltitrexed (trade name Tomudex). Five agentswere in clinical trials in 2002: raltitrexed, pemetrexed, nolatrexed,ZD9331, and GS7904L. Additional non-limiting examples include:Raltitrexed, used for colorectal cancer since 1998; Fluorouracil, usedfor colorectal cancer; BGC 945; OSI-7904L. In some embodiments, thistreatment regimen is for advanced cancer. In some embodiments, thistreatment regimen is excluded for early cancer.

In further embodiments, there may be involved prodrugs that can beconverted to thymidylate synthase inhibitors in the body, such asCapecitabine (INN), an orally-administered chemotherapeutic agent usedin the treatment of numerous cancers. Capecitabine is a prodrug, that isenzymatically converted to 5-fluorouracil in the body. In someembodiments, this treatment regimen is for advanced cancer. In someembodiments, this treatment regimen is excluded for early cancer.

If cancer has entered the lymph nodes, adding the chemotherapy agentsfluorouracil or capecitabine increases life expectancy. If the lymphnodes do not contain cancer, the benefits of chemotherapy arecontroversial. If the cancer is widely metastatic or unresectable,treatment is then palliative. For example, a number of differentchemotherapy medications may be used. Chemotherapy agents for thiscondition may include capecitabine, fluorouracil, irinotecan,leucovorin, oxaliplatin and UFT. Another type of agent that is sometimesused are the epidermal growth factor receptor inhibitors. In someembodiments, this treatment regimen is for advanced cancer. In someembodiments, this treatment regimen is excluded for early cancer.

In certain embodiments, alternative treatments may be prescribed orrecommended based on the biomarker profile. In addition to traditionalchemotherapy for colorectal cancer patients, cancer therapies alsoinclude a variety of combination therapies with both chemical andradiation based treatments. Combination chemotherapies include, forexample, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen,raloxifene, estrogen receptor binding agents, taxol, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum,5-fluorouracil, vincristin, vinblastin and methotrexate, or any analogor derivative variant of the foregoing. In some embodiments, treatmentwith one or more of the compounds described herein is for advancedcancer. In some embodiments, treatment with one or more of the compoundsdescribed herein is excluded for early cancer.

While a combination of radiation and chemotherapy may be useful forrectal cancer, its use in colon cancer is not routine due to thesensitivity of the bowels to radiation. Just as for chemotherapy,radiotherapy can be used in the neoadjuvant and adjuvant setting forsome stages of rectal cancer. In some embodiments, this treatmentregimen is for advanced cancer. In some embodiments, this treatmentregimen is excluded for early cancer.

In people with incurable colorectal cancer, treatment options includingpalliative care can be considered for improving quality of life.Surgical options may include non-curative surgical removal of some ofthe cancer tissue, bypassing part of the intestines, or stent placement.These procedures can be considered to improve symptoms and reducecomplications such as bleeding from the tumor, abdominal pain andintestinal obstruction. Non-operative methods of symptomatic treatmentinclude radiation therapy to decrease tumor size as well as painmedications. In some embodiments, this treatment regimen is for advancedcancer. In some embodiments, this treatment regimen is excluded forearly cancer.

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells. In some embodiments, this treatmentregimen is for advanced cancer. In some embodiments, this treatmentregimen is excluded for early cancer.

Generally, the tumor cell must bear some marker that is amenable totargeting, i.e., is not present on the majority of other cells. Manytumor markers exist and any of these may be suitable for targeting.Common tumor markers include carcinoembryonic antigen, prostate specificantigen, urinary tumor associated antigen, fetal antigen, tyrosinase(p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP,estrogen receptor, laminin receptor, erb B and p155. Markers describedherein may be used in the context of the current claims for the purposesof developing a targeting moiety. For example, the targeting moiety maybe one that binds the tumor marker. In some embodiments, the targetingmoiety is an antibody. In further embodiments, the targeting moiety isan aptamer or aptamir.

In yet another embodiment, the treatment is a gene therapy. In certainembodiments, the therapeutic gene is a tumor suppressor gene. A tumorsuppressor gene is a gene that, when present in a cell, reduces thetumorigenicity, malignancy, or hyperproliferative phenotype of the cell.This definition includes both the full length nucleic acid sequence ofthe tumor suppressor gene, as well as non-full length sequences of anylength derived from the full length sequences. It being furtherunderstood that the sequence includes the degenerate codons of thenative sequence or sequences which may be introduced to provide codonpreference in a specific host cell. Examples of tumor suppressor nucleicacids within this definition include, but are not limited to APC, CYLD,HIN-I, KRAS2b, pló, p19, p21, p27, p27mt, p53, p57, p73, PTEN, Rb,Uteroglobin, Skp2, BRCA-I, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2/JV18,MEN1, MEN2, MTS1, NF1, NF2, VHL, WRN, WT1, CFTR, C-CAM, CTS-I, zacl,scFV, MMAC1, FCC, MCC, Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), 101F6, Gene 21 (NPRL2), or agene encoding a SEM A3 polypeptide and FUS1. Other exemplary tumorsuppressor genes are described in a database of tumor suppressor genesat www.cise.ufl.edu/˜yyl/HTML-TSGDB/Homepage.litml. This database isherein specifically incorporated by reference into this and all othersections of the present application. Nucleic acids encoding tumorsuppressor genes, as discussed above, include tumor suppressor genes, ornucleic acids derived therefrom (e.g., cDNAs, cRNAs, mRNAs, andsubsequences thereof encoding active fragments of the respective tumorsuppressor amino acid sequences), as well as vectors comprising thesesequences. One of ordinary skill in the art would be familiar with tumorsuppressor genes that can be applied.

C. Monitoring

In certain aspects, the biomarker-based method may be combined with oneor more other colon cancer diagnosis or screening tests at increasedfrequency if the patient is determined to be at high risk for recurrenceor have a poor prognosis based on the biomarker described above.

The colon monitoring may include any methods known in the art. Inparticular, the monitoring include obtaining a sample and testing thesample for diagnosis. For example, the colon monitoring may includecolonoscopy or coloscopy, which is the endoscopic examination of thelarge bowel and the distal part of the small bowel with a CCD camera ora fiber optic camera on a flexible tube passed through the anus. It canprovide a visual diagnosis (e.g. ulceration, polyps) and grants theopportunity for biopsy or removal of suspected colorectal cancerlesions. Thus, colonoscopy or coloscopy can be used for treatment.

In further aspects, the monitoring diagnosis may include sigmoidoscopy,which is similar to colonoscopy—the difference being related to whichparts of the colon each can examine. A colonoscopy allows an examinationof the entire colon (1200-1500 mm in length). A sigmoidoscopy allows anexamination of the distal portion (about 600 mm) of the colon, which maybe sufficient because benefits to cancer survival of colonoscopy havebeen limited to the detection of lesions in the distal portion of thecolon. A sigmoidoscopy is often used as a screening procedure for a fullcolonoscopy, often done in conjunction with a fecal occult blood test(FOBT). About 5% of these screened patients are referred to colonoscopy.

In additional aspects, the monitoring diagnosis may include virtualcolonoscopy, which uses 2D and 3D imagery reconstructed from computedtomography (CT) scans or from nuclear magnetic resonance (MR) scans, asa totally non-invasive medical test.

The monitoring include the use of one or more screening tests for coloncancer including, but not limited to fecal occult blood testing,flexible sigmoidoscopy and colonoscopy. Of the three, only sigmoidoscopycannot screen the right side of the colon where 42% of malignancies arefound. Virtual colonoscopy via a CT scan appears as good as standardcolonoscopy for detecting cancers and large adenomas but is expensive,associated with radiation exposure, and cannot remove any detectedabnormal growths like standard colonoscopy can. Fecal occult bloodtesting (FOBT) of the stool is typically recommended every two years andcan be either guaiac based or immunochemical. Annual FOBT screeningresults in a 16% relative risk reduction in colorectal cancer mortality,but no difference in all-cause mortality. The M2-PK test identifies anenzyme in colorectal cancers and polyps rather than blood in the stool.It does not require any special preparation prior to testing. M2-PK issensitive for colorectal cancer and polyps and is able to detectbleeding and non-bleeding colorectal cancer and polyps. In the event ofa positive result people would be asked to undergo further examinatione.g. colonoscopy.

D. ROC Analysis

In statistics, a receiver operating characteristic (ROC), or ROC curve,is a graphical plot that illustrates the performance of a binaryclassifier system as its discrimination threshold is varied. The curveis created by plotting the true positive rate against the false positiverate at various threshold settings. (The true-positive rate is alsoknown as sensitivity in biomedical informatics, or recall in machinelearning. The false-positive rate is also known as the fall-out and canbe calculated as 1−specificity). The ROC curve is thus the sensitivityas a function of fall-out. In general, if the probability distributionsfor both detection and false alarm are known, the ROC curve can begenerated by plotting the cumulative distribution function (area underthe probability distribution from −infinity to +infinity) of thedetection probability in the y-axis versus the cumulative distributionfunction of the false-alarm probability in x-axis.

ROC analysis provides tools to select possibly optimal models and todiscard suboptimal ones independently from (and prior to specifying) thecost context or the class distribution. ROC analysis is related in adirect and natural way to cost/benefit analysis of diagnostic decisionmaking.

The ROC curve was first developed by electrical engineers and radarengineers during World War II for detecting enemy objects inbattlefields and was soon introduced to psychology to account forperceptual detection of stimuli. ROC analysis since then has been usedin medicine, radiology, biometrics, and other areas for many decades andis increasingly used in machine learning and data mining research.

The ROC is also known as a relative operating characteristic curve,because it is a comparison of two operating characteristics (TPR andFPR) as the criterion changes. ROC analysis curves are known in the artand described in Metz C E (1978) Basic principles of ROC analysis.Seminars in Nuclear Medicine 8:283-298; Youden W J (1950) An index forrating diagnostic tests. Cancer 3:32-35; Zweig M H, Campbell G (1993)Receiver-operating characteristic (ROC) plots: a fundamental evaluationtool in clinical medicine. Clinical Chemistry 39:561-577; and Greiner M,Pfeiffer D, Smith R D (2000) Principles and practical application of thereceiver-operating characteristic analysis for diagnostic tests.Preventive Veterinary Medicine 45:23-41, which are herein incorporatedby reference in their entirety.

III. SAMPLE PREPARATION

In certain aspects, methods involve obtaining a sample from a subject.The methods of obtaining provided herein may include methods of biopsysuch as fine needle aspiration, core needle biopsy, vacuum assistedbiopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsyor skin biopsy. In certain embodiments the sample is obtained from abiopsy from colorectal tissue by any of the biopsy methods previouslymentioned. In other embodiments the sample may be obtained from any ofthe tissues provided herein that include but are not limited tonon-cancerous or cancerous tissue and non-cancerous or cancerous tissuefrom the serum, gall bladder, mucosal, skin, heart, lung, breast,pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon,intestine, brain, prostate, esophagus, or thyroid tissue. Alternatively,the sample may be obtained from any other source including but notlimited to blood, sweat, hair follicle, buccal tissue, tears, menses,feces, or saliva. In certain aspects the sample is obtained from cysticfluid or fluid derived from a tumor or neoplasm. In yet otherembodiments the cyst, tumor or neoplasm is colorectal. In certainaspects of the current methods, any medical professional such as adoctor, nurse or medical technician may obtain a biological sample fortesting. Yet further, the biological sample can be obtained without theassistance of a medical professional.

A sample may include but is not limited to, tissue, cells, or biologicalmaterial from cells or derived from cells of a subject. The biologicalsample may be a heterogeneous or homogeneous population of cells ortissues. The biological sample may be obtained using any method known tothe art that can provide a sample suitable for the analytical methodsdescribed herein. The sample may be obtained by non-invasive methodsincluding but not limited to: scraping of the skin or cervix, swabbingof the cheek, saliva collection, urine collection, feces collection,collection of menses, tears, or semen.

The sample may be obtained by methods known in the art. In certainembodiments the samples are obtained by biopsy. In other embodiments thesample is obtained by swabbing, scraping, phlebotomy, or any othermethods known in the art. In some cases, the sample may be obtained,stored, or transported using components of a kit of the present methods.In some cases, multiple samples, such as multiple colorectal samples maybe obtained for diagnosis by the methods described herein. In othercases, multiple samples, such as one or more samples from one tissuetype (for example colon) and one or more samples from another tissue(for example buccal) may be obtained for diagnosis by the methods. Insome cases, multiple samples such as one or more samples from one tissuetype (e.g. rectal) and one or more samples from another tissue (e.g.cecum) may be obtained at the same or different times. Samples may beobtained at different times are stored and/or analyzed by differentmethods. For example, a sample may be obtained and analyzed by routinestaining methods or any other cytological analysis methods.

In some embodiments the biological sample may be obtained by aphysician, nurse, or other medical professional such as a medicaltechnician, endocrinologist, cytologist, phlebotomist, radiologist, or apulmonologist. The medical professional may indicate the appropriatetest or assay to perform on the sample. In certain aspects a molecularprofiling business may consult on which assays or tests are mostappropriately indicated. In further aspects of the current methods, thepatient or subject may obtain a biological sample for testing withoutthe assistance of a medical professional, such as obtaining a wholeblood sample, a urine sample, a fecal sample, a buccal sample, or asaliva sample.

In other cases, the sample is obtained by an invasive procedureincluding but not limited to: biopsy, needle aspiration, or phlebotomy.The method of needle aspiration may further include fine needleaspiration, core needle biopsy, vacuum assisted biopsy, or large corebiopsy. In some embodiments, multiple samples may be obtained by themethods herein to ensure a sufficient amount of biological material.

General methods for obtaining biological samples are also known in theart. Publications such as Ramzy, Ibrahim Clinical Cytopathology andAspiration Biopsy 2001, which is herein incorporated by reference in itsentirety, describes general methods for biopsy and cytological methods.In one embodiment, the sample is a fine needle aspirate of a colorectalor a suspected colorectal tumor or neoplasm. In some cases, the fineneedle aspirate sampling procedure may be guided by the use of anultrasound, X-ray, or other imaging device.

In some embodiments of the present methods, the molecular profilingbusiness may obtain the biological sample from a subject directly, froma medical professional, from a third party, or from a kit provided by amolecular profiling business or a third party. In some cases, thebiological sample may be obtained by the molecular profiling businessafter the subject, a medical professional, or a third party acquires andsends the biological sample to the molecular profiling business. In somecases, the molecular profiling business may provide suitable containers,and excipients for storage and transport of the biological sample to themolecular profiling business.

In some embodiments of the methods described herein, a medicalprofessional need not be involved in the initial diagnosis or sampleacquisition. An individual may alternatively obtain a sample through theuse of an over the counter (OTC) kit. An OTC kit may contain a means forobtaining said sample as described herein, a means for storing saidsample for inspection, and instructions for proper use of the kit. Insome cases, molecular profiling services are included in the price forpurchase of the kit. In other cases, the molecular profiling servicesare billed separately. A sample suitable for use by the molecularprofiling business may be any material containing tissues, cells,nucleic acids, proteins, polypeptides, genes, gene fragments, expressionproducts, gene expression products, protein expression products orfragments, or gene expression product fragments of an individual to betested. Methods for determining sample suitability and/or adequacy areprovided.

In some embodiments, the subject may be referred to a specialist such asan oncologist, surgeon, or endocrinologist. The specialist may likewiseobtain a biological sample for testing or refer the individual to atesting center or laboratory for submission of the biological sample. Insome cases the medical professional may refer the subject to a testingcenter or laboratory for submission of the biological sample. In othercases, the subject may provide the sample. In some cases, a molecularprofiling business may obtain the sample.

IV. NUCLEIC ACID ASSAYS

Aspects of the methods include assaying nucleic acids to determineexpression or activity levels. Arrays can be used to detect differencesbetween two samples. Specifically contemplated applications includeidentifying and/or quantifying differences between RNA from a samplethat is normal and from a sample that is not normal, between a cancerouscondition and a non-cancerous condition, or between two differentlytreated samples. Also, RNA may be compared between a sample believed tobe susceptible to a particular disease or condition and one believed tobe not susceptible or resistant to that disease or condition. A samplethat is not normal is one exhibiting phenotypic trait(s) of a disease orcondition or one believed to be not normal with respect to that diseaseor condition. It may be compared to a cell that is normal with respectto that disease or condition. Phenotypic traits include symptoms of, orsusceptibility to, a disease or condition of which a component is or mayor may not be genetic or caused by a hyperproliferative or neoplasticcell or cells.

An array comprises a solid support with nucleic acid probes attached tothe support. Arrays typically comprise a plurality of different nucleicacid probes that are coupled to a surface of a substrate in different,known locations. These arrays, also described as “microarrays” orcolloquially “chips” have been generally described in the art, forexample, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195,6,040,193, 5,424,186 and Fodor et al., 1991), each of which isincorporated by reference in its entirety for all purposes. Techniquesfor the synthesis of these arrays using mechanical synthesis methods aredescribed in, e.g., U.S. Pat. No. 5,384,261, incorporated herein byreference in its entirety for all purposes. Although a planar arraysurface is used in certain aspects, the array may be fabricated on asurface of virtually any shape or even a multiplicity of surfaces.Arrays may be nucleic acids on beads, gels, polymeric surfaces, fiberssuch as fiber optics, glass or any other appropriate substrate, see U.S.Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992,which are hereby incorporated in their entirety for all purposes.

In addition to the use of arrays and microarrays, it is contemplatedthat a number of difference assays could be employed to analyze nucleicacids, their activities, and their effects. Such assays include, but arenot limited to, nucleic amplification, polymerase chain reaction,quantitative PCR, RT-PCR, in situ hybridization, Northern hybridization,hybridization protection assay (HPA)(GenProbe), branched DNA (bDNA)assay (Chiron), rolling circle amplification (RCA), single moleculehybridization detection (US Genomics), Invader assay (ThirdWaveTechnologies), and/or Bridge Litigation Assay (Genaco).

A further assay useful for quantifying and/or identifying nucleic acidsis RNAseq. RNA-seq (RNA sequencing), also called whole transcriptomeshotgun sequencing, uses next-generation sequencing (NGS) to reveal thepresence and quantity of RNA in a biological sample at a given moment intime. RNA-Seq is used to analyze the continually changing cellulartranscriptome. Specifically, RNA-Seq facilitates the ability to look atalternative gene spliced transcripts, post-transcriptionalmodifications, gene fusion, mutations/SNPs and changes in geneexpression. In addition to mRNA transcripts, RNA-Seq can look atdifferent populations of RNA to include total RNA, small RNA, such asmiRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used todetermine exon/intron boundaries and verify or amend previouslyannotated 5′ and 3′ gene boundaries.

V. PROTEIN ASSAYS

A variety of techniques can be employed to measure expression levels ofpolypeptides and proteins in a biological sample. Examples of suchformats include, but are not limited to, enzyme immunoassay (EIA),radioimmunoassay (RIA), Western blot analysis and enzyme linkedimmunoabsorbant assay (ELISA). A skilled artisan can readily adapt knownprotein/antibody detection methods for use in determining proteinexpression levels of biomarkers.

In one embodiment, antibodies, or antibody fragments or derivatives, canbe used in methods such as Western blots or immunofluorescencetechniques to detect biomarker expression. In some embodiments, eitherthe antibodies or proteins are immobilized on a solid support. Suitablesolid phase supports or carriers include any support capable of bindingan antigen or an antibody. Well-known supports or carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite.

One skilled in the art will know many other suitable carriers forbinding antibody or antigen, and will be able to adapt such support foruse with the present disclosure. The support can then be washed withsuitable buffers followed by treatment with the detectably labeledantibody. The solid phase support can then be washed with the buffer asecond time to remove unbound antibody. The amount of bound label on thesolid support can then be detected by conventional means.

Immunohistochemistry methods are also suitable for detecting theexpression levels of biomarkers. In some embodiments, antibodies orantisera, including polyclonal antisera, and monoclonal antibodiesspecific for each marker may be used to detect expression. Theantibodies can be detected by direct labeling of the antibodiesthemselves, for example, with radioactive labels, fluorescent labels,hapten labels such as, biotin, or an enzyme such as horse radishperoxidase or alkaline phosphatase. Alternatively, unlabeled primaryantibody is used in conjunction with a labeled secondary antibody,comprising antisera, polyclonal antisera or a monoclonal antibodyspecific for the primary antibody. Immunohistochemistry protocols andkits are well known in the art and are commercially available.

Immunological methods for detecting and measuring complex formation as ameasure of protein expression using either specific polyclonal ormonoclonal antibodies are known in the art. Examples of such techniquesinclude enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays(RIAs), fluorescence-activated cell sorting (FACS) and antibody arrays.Such immunoassays typically involve the measurement of complex formationbetween the protein and its specific antibody. These assays and theirquantitation against purified, labeled standards are well known in theart. A two-site, monoclonal-based immunoassay utilizing antibodiesreactive to two non-interfering epitopes or a competitive binding assaymay be employed.

Numerous labels are available and commonly known in the art.Radioisotope labels include, for example, ³⁶S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I.The antibody can be labeled with the radioisotope using the techniquesknown in the art. Fluorescent labels include, for example, labels suchas rare earth chelates (europium chelates) or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, Lissamine,phycoerythrin and Texas Red are available. The fluorescent labels can beconjugated to the antibody variant using the techniques known in theart. Fluorescence can be quantified using a fluorimeter. Variousenzyme-substrate labels are available and U.S. Pat. Nos. 4,275,149,4,318,980 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,.beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for Use inEnzyme Immunoassay, in Methods in Enzymology (Ed. J. Langone & H. VanVunakis), Academic press, New York, 73: 147-166 (1981).

In some embodiments, a detection label is indirectly conjugated with anantibody. The skilled artisan will be aware of various techniques forachieving this. For example, the antibody can be conjugated with biotinand any of the three broad categories of labels mentioned above can beconjugated with avidin, or vice versa. Biotin binds selectively toavidin and thus, the label can be conjugated with the antibody in thisindirect manner. Alternatively, to achieve indirect conjugation of thelabel with the antibody, the antibody is conjugated with a small hapten(e.g., digoxin) and one of the different types of labels mentioned aboveis conjugated with an anti-hapten antibody (e.g., anti-digoxinantibody). In some embodiments, the antibody need not be labeled, andthe presence thereof can be detected using a labeled antibody, whichbinds to the antibody.

VI. PHARMACEUTICAL COMPOSITIONS

In certain aspects, the compositions or agents for use in the methods,such as chemotherapeutic agents or biomarker modulators, are suitablycontained in a pharmaceutically acceptable carrier. The carrier isnon-toxic, biocompatible and is selected so as not to detrimentallyaffect the biological activity of the agent. The agents in some aspectsof the disclosure may be formulated into preparations for local delivery(i.e. to a specific location of the body, such as skeletal muscle orother tissue) or systemic delivery, in solid, semi-solid, gel, liquid orgaseous forms such as tablets, capsules, powders, granules, ointments,solutions, depositories, inhalants and injections allowing for oral,parenteral or surgical administration. Certain aspects of the disclosurealso contemplate local administration of the compositions by coatingmedical devices and the like.

Suitable carriers for parenteral delivery via injectable, infusion orirrigation and topical delivery include distilled water, physiologicalphosphate-buffered saline, normal or lactated Ringer's solutions,dextrose solution, Hank's solution, or propanediol. In addition,sterile, fixed oils may be employed as a solvent or suspending medium.For this purpose any biocompatible oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables. The carrier and agentmay be compounded as a liquid, suspension, polymerizable ornon-polymerizable gel, paste or salve.

The carrier may also comprise a delivery vehicle to sustain (i.e.,extend, delay or regulate) the delivery of the agent(s) or to enhancethe delivery, uptake, stability or pharmacokinetics of the therapeuticagent(s). Such a delivery vehicle may include, by way of non-limitingexamples, microparticles, microspheres, nanospheres or nanoparticlescomposed of proteins, liposomes, carbohydrates, synthetic organiccompounds, inorganic compounds, polymeric or copolymeric hydrogels andpolymeric micelles.

In certain aspects, the actual dosage amount of a compositionadministered to a patient or subject can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active agent, such as an isolatedexosome, a related lipid nanovesicle, or an exosome or nanovesicleloaded with therapeutic agents or diagnostic agents. In otherembodiments, the active agent may comprise between about 2% to about 75%of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 microgram/kg/body weight to about 100mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered.

Solutions of pharmaceutical compositions can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions also can be prepared in glycerol, liquid polyethyleneglycols, mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

In certain aspects, the pharmaceutical compositions are advantageouslyadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsified. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain 10 mg or less, 25 mg, 50 mg or up to about100 mg of human serum albumin per milliliter of phosphate bufferedsaline. Other pharmaceutically acceptable carriers include aqueoussolutions, non-toxic excipients, including salts, preservatives, buffersand the like.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate.Aqueous carriers include water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial agents, antgifungalagents, anti-oxidants, chelating agents and inert gases. The pH andexact concentration of the various components the pharmaceuticalcomposition are adjusted according to well-known parameters.

Additional formulations are suitable for oral administration. Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders.

In further aspects, the pharmaceutical compositions may include classicpharmaceutical preparations. Administration of pharmaceuticalcompositions according to certain aspects may be via any common route solong as the target tissue is available via that route. This may includeoral, nasal, buccal, rectal, vaginal or topical. Topical administrationmay be particularly advantageous for the treatment of skin cancers, toprevent chemotherapy-induced alopecia or other dermal hyperproliferativedisorder. Alternatively, administration may be by orthotopic,intradermal, subcutaneous, intramuscular, intraperitoneal or intravenousinjection. Such compositions would normally be administered aspharmaceutically acceptable compositions that include physiologicallyacceptable carriers, buffers or other excipients. For treatment ofconditions of the lungs, aerosol delivery can be used. Volume of theaerosol is between about 0.01 ml and 0.5 ml.

An effective amount of the pharmaceutical composition is determinedbased on the intended goal. The term “unit dose” or “dosage” refers tophysically discrete units suitable for use in a subject, each unitcontaining a predetermined-quantity of the pharmaceutical compositioncalculated to produce the desired responses discussed above inassociation with its administration, i.e., the appropriate route andtreatment regimen. The quantity to be administered, both according tonumber of treatments and unit dose, depends on the protection or effectdesired.

Precise amounts of the pharmaceutical composition also depend on thejudgment of the practitioner and are peculiar to each individual.Factors affecting the dose include the physical and clinical state ofthe patient, the route of administration, the intended goal of treatment(e.g., alleviation of symptoms versus cure) and the potency, stabilityand toxicity of the particular therapeutic substance.

VII. KITS

Certain aspects of the present disclosure also concern kits containingcompositions of the disclosure or compositions to implement methods ofthe disclosure. In some embodiments, kits can be used to evaluate one ormore nucleic acid and/or polypeptide molecules. In certain embodiments,a kit contains, contains at least or contains at most 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more nucleic acid probes,synthetic RNA molecules or inhibitors, or any value or range andcombination derivable therein. In some embodiments, there are kits forevaluating biomarker levels or activity in a cell.

Kits may comprise components, which may be individually packaged orplaced in a container, such as a tube, bottle, vial, syringe, or othersuitable container means.

Individual components may also be provided in a kit in concentratedamounts; in some embodiments, a component is provided individually inthe same concentration as it would be in a solution with othercomponents. Concentrations of components may be provided as 1×, 2×, 5×,10×, or 20× or more.

Kits for using probes, polypeptide detecting agents, and/or inhibitorsor antagonists of the disclosure for prognostic or diagnosticapplications are included. Specifically contemplated are any suchmolecules corresponding to any biomarker nucleic acid or polypeptide.

In certain aspects, negative and/or positive control agents are includedin some kit embodiments. The control molecules can be used to verifytransfection efficiency and/or control for transfection-induced changesin cells.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein and that different embodiments may be combined.

Any embodiment of the disclosure relating to a polypeptide or nucleicacid is contemplated also to cover embodiments whose sequences are atleast 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99% identical to the polypeptide or nucleic acid.

Embodiments of the disclosure include kits for analysis of apathological sample by assessing a nucleic acid or polypeptide profilefor a sample comprising, in suitable container means, two or more RNAprobes, or a biomarker polypeptide detecting agent, wherein the RNAprobes or polypeptide detecting agent detects biomarker nucleic acids orpolypeptides. In some embodiments, the reagents (i.e. RNA probe and/orpolypeptide detecting agent) are labeled with a detectable label. Labelsare known in the art and also described herein. The kit can furthercomprise reagents for labeling probes, nucleic acids, and/or detectingagents. The kit may also include labeling reagents, including at leastone of amine-modified nucleotide, poly(A) polymerase, and poly(A)polymerase buffer. Labeling reagents can include an amine-reactive dye.

VIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the disclosure, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe disclosure.

Example 1—Identification of a miRNA Cluster with Prognostic BiomarkerPotential in Colorectal Cancer

Despite recent advances in colorectal cancer (CRC) treatments,approximately 30-50% of CRC patients who undergo curative resectionsubsequently experience tumor recurrence. Therefore, identification ofreliable prognostic biomarkers that accurately select and stratifyhigh-risk stage II & III CRC patients is paramount for individualizedtherapeutic strategies and ultimately improving patient outcomes.MicroRNAs (miRNAs) are small non-coding RNAs frequently dysregulated invarious cancers and are emerging as promising cancer biomarkers. In thisexample, the inventors conducted a comprehensive miRNA expressionprofiling of tumors with or without tumor recurrence to identify a panelof miRNAs that are associated with tumor recurrence in CRC patients.

The inventors performed miRNA expression profiling in stage III primaryCRC using multiple datasets to characterize miRNA signature and identifytargets in patients with and without tumor recurrence (FIG. 1). Theexpression of these candidate miRNAs was validated in two independentstage II & III CRC cohorts (n=88, 107). The expression of the candidatetissue-based miRNAs were evaluated in 119 plasma specimens from patientswith stage II/III CRC.

The inventors identified that 12% of differentially expressed miRNAs intumor tissues from recurrence-positive CRC patients were clusteredtogether and encoded by 14q32 locus. Intriguingly, all of these miRNAswere overexpressed in the tumor tissues with recurrence. Usingbioinformatic and statistical approaches six differentially expressedmiRNAs that mapped to this 14q32 locus were identified, and theoverexpression of three miRNAs (miR-409, -432 and -758) was confirmed inthe tumor tissues from stage II & III CRC patients with vs. withoutrelapse (all p<0.05) (FIG. 1D, 2A). Furthermore, high expression ofmiR-409, miR-432 and miR-758 was associated with poor disease-freesurvival (DFS) (p<0.05, p<0.05 and p<0.001 respectively) (FIG. 2B). Theinventors thereafter validated the prognostic potential of miR-758 in anindependent patient cohort, and Cox regression analysis revealed thatoverexpression of miR-758 emerged as an independent prognostic factorfor DFS (HR:2.2, 95% CI:1.0-4.9, p<0.05). Interestingly, the expressionof circulating miR-758 was significantly higher in patients with vs.without recurrence in pre-operative serum samples (p<0.01).

Using a comprehensive miRNA expression profiling approach, the inventorshave identified a cluster of miRNAs encoded at 14q32, which isfrequently overexpressed in CRC patients with tumor recurrence. Inparticular, miR-758 was able to identify a subgroup of stage II/IIIpatients with high-risk of recurrence and was upregulated inpost-operative plasma samples of recurrence-positive CRC patients (FIG.3), suggesting its potential usefulness as an important biomarker foridentification of high-risk patients that are candidates for moreaggressive chemotherapy.

Example 2—Genome-Wide Discovery and Identification of a Novel microRNASignature for Recurrence Prediction in Colorectal Cancer

Colorectal cancer (CRC) is one of the leading causes of cancer-relateddeaths worldwide. In 2016, there were estimated 95,270 newly diagnosedCRC cases, and 49,190 deaths from this disease in the United States.Survival of patients is closely associated with the tumor stage at thetime of diagnosis as 5-year relative survival rates range from 65% forall stages, 90% for localized primary tumor, 71% for regional metastasisand 13% for distant metastasis. Post-surgery, adjuvant therapy is onlyrecommended to those with high risk stage II, as well as stage III andIV tumors. However, approximately 40-50% of the patients undergocurative surgery only and 20-30% that are treated with adjuvantchemotherapy, eventually relapse and experience a metastatic disease andeventual death. The current gold standard TNM (Tumor, Node, Metastasis)staging for determining the prognosis of CRC patients remains inadequateat identification of high-risk stage II and III patients that have ahigh potential of developing tumor recurrence. MicroRNAs (miRNAs) playan important role in CRC development and are emerging as importantdisease biomarkers. Therefore in this example, the inventors sought todetermine the prognostic potential of the miRNAs using a systematic,genome-wide biomarker discovery approach, followed by validation ofbiomarkers in multiple patient cohorts

Three independent publicly available genome-wide miRNA expressiondatasets were used for miRNA biomarker discovery (n=158) and in-silicovalidation (n=109 and n=40) of this miRNA signature for recurrenceprediction in stage II and III CRC using Cox's regression. The eightgene miRNA signature discovered from the genome-wide analysis wasanalytically validated in two independent patient cohorts (n=127 andn=96) using Taqman-based RT-PCR assays (FIG. 5).

The genome-wide comprehensive analysis led to the identification of aneight gene miRNA classifier that significantly predicted recurrence freesurvival (RFS) in training (log rank p=0.003) and two independentvalidation cohorts (log rank p<0.0001 and p=0.002). The RT-PCR basedtraining and validation of the eight gene classifier in two independentclinical cohorts significantly associated with poor prognosis in stageII and III CRC patients (log rank p<0.004 and p<0.0001). Multivariateanalyses performed in these two patient cohorts revealed that the eightgene miRNA classifier served as an independent predictor of poorprognosis in stage II and III CRC patients (FIG. 6-7).

In conclusion, the inventors have identified a novel miRNA-basedclassifier, which is robustly predictive of poor prognosis in patentswith stage II/III CRC, and might facilitate identification andstratification of high-risk patients that are candidates for adjuvantchemotherapy and clinical surveillance.

Example 3—a Novel miRNA Signature for the Detection of Lymph NodeMetastasis in Submucosal Colorectal Cancer Patients

Owing to recent advances in colonoscopic techniques, majority ofsubmucosal colorectal cancers (T1 CRCs) can now be removed by endoscopicresection. Among these, 70% of T1 CRCs are deemed as “high risk” becausethey meet certain pathological risk factors including the presence oflymphovascular invasion, poor differentiation, and increased depth oftumor (>1000 um). However, post-surgical pathology data indicates thatin reality only 10-15% of all T1 CRCs are genuinely high risk, while allother patients undergo unnecessary surgeries. Since current pathologicalcriteria are inadequate, availability of more robust molecularbiomarkers that may help identify ‘genuine high risk patients with lymphnode (LN) metastasis’ more accurately will reduce the burden of surgicalovertreatments. Due to the growing interest in developing miRNAbiomarkers, the inventors undertook this study to identify a miRNA-baseddiagnostic signature for detecting LN metastasis in CRC.

In a biomarker discovery step using RNA-Seq data from 15 LN-positive and104 LN-negative T½ CRC patients, the inventors identified candidatemiRNAs with >0.5 log fold change and a p<0.05. Thereafter, using areceiver operating curve (ROC) based backwards elimination approach, theinventors identified a signature of miRNAs that were differentiallyexpresses in LN positive vs. negative CRCs. The inventors validated theperformance of this miRNA signature to detect LN metastasis in 190surgically resected CRC specimens from two independent patient cohortsby qPCR assays.

A panel of 10 differentially expressed miRNAs were identified in thediscovery step, which was initially validated in a training cohort of 61T1 CRC samples, which included 8 LN-positive cases. Using a logisticregression analysis model, the inventors deduced robust AUC values whenusing miRNA expression results alone (AUC=0.85, 95% CI: 0.74-0.93,p<0.001) for identifying LN-positive T1 CRCs. Thereafter using the samemodel parameters in an independent validation cohort of 130 T1 CRCs,which included 16 LN-positive patients, the inventors were able tosuccessfully confirm the results from the training cohort (AUC=0.74, 95%CI:0.66-0.781, p<0.001) for the identification of high risk T1 CRCpatients with lymph node metastasis.

Based upon a systematic approach, this example demonstrates report thefeasibility and promise of a miRNA signature that can be used clinicallyfor the detection of T1 CRCs with lymph node metastasis, which willreduce patient discomfort and healthcare costs.

A. Patients and Methods

1. Candidate miRNA Selection and miRNA Signature Construction

The inventors analyzed and constructed miRNA signature using miRNA seqof TCGA database. First, the inventors compared miRNA expressionsbetween LNM positive and negative samples in 119 T1-2 CRC (positive 15and negative 104, respectively) since T1 there were only 19 CRC sampleswith node information (positive 2 and negative 17, respectively). TCGAmiRNA seq included 1881 miRNAs and candidate miRNAs were selectedthrough the following analyses; (1) log 2 fold change of >0.5 betweennode positive and negative samples with p-value of Wilcoxon test <0.05and (2) The average expression of >3. Then receiver operation curve(ROC) based back step elimination method was applied to Construct themiRNA Signature by Logistic Regression Model.

2. Patients and sample collection

Total, 188 T1 CRC fresh frozen paraffin embedded (FFPE) specimens wereobtained from two Japanese cohorts: 60 (LNM positive 7 and negative 53,respectively) from Kumamoto University cohort and 128 (LNM positive 20and negative 108, respectively) from the University of Tokyo cohort. Allof the samples were resected surgically and collected between 2005 and2014 in Kumamoto University and between 2005 and 2016 in the Universityof Tokyo. Matched biopsy samples (FFPE), which were taken duringcolonoscopy before surgery, were also obtained from the University ofTokyo cohort. Exclusion criteria were as follows: (1) synchronous CRC(T1-4); (2) distant metastasis; (3) neoadjuvant chemo/radio therapy; (4)hereditary or inflammation associated CRC; and (5) non-adenocarcinoma.All of the patients underwent standard surgical procedure (resection ofaffected segment of colon or rectum and regional lymphadenectomy), andall of the samples were evaluated by pathologists of each instituteaccording to AJCC TNM grading system and Japanese guidelines.Preoperative serum carcinoembrionic antigen (CEA) level was evaluatedbefore surgery in the laboratory. CT were performed before surgery forall of the patients of the University of Tokyo cohort and their findingswere evaluated by radiologists in the University of Tokyo hospital. Whenthe size of regional LN is more than 10 mm, LNM was estimated aspositive.

3. Total RNA Extraction and Real-Time Quantitative Reverse TranscriptionPolymerase Chain Reaction

Total RNA was extracted from 10 μm thick FFPE specimens using AllPrep®DNA/RNA FFPE kit (QIAGE, Hilden, Germany) according to themanufacturer's instructions. cDNA was synthesized from total RNAaccording to the manufacturer's recommendations (ThermoFisherScientific, MA, USA). qRT-PCR was performed using the QuantStudio™ 7Flex Real Time PCR System (Applied Biosystems®, Foster City, Calif.),and expression levels were evaluated with Applied BiosystemsQuantStudio™ 7 Flex Real Time PCR System Software. The relativeabundance of target transcripts was evaluated and normalized to theexpression levels of miR-16 as internal controls using the 2^(−ΔCt)method; ΔCt means the difference of Ct values between the miRNA ofinterest and the normalizer. Normalized values were further logtransformed and standardized. All of the primers for miRNA used in thisstudy are purchased from ThermoFisher Scientific (MA, USA).

4. Study Design

miRNA signatures to predict LNM in T1-2 CRC, which was constructed usingTCGA database, were validated by in silico analyses of T1 CRC patientsof TCGA database and also GSE56350 which included miRNA expression of 46primary site and 43 LNM site evaluated by Affymetrix microarray.

Then, the inventors validated the predictive power of this miRNAsignature in surgically resected T1 CRC FFPE samples from two individualcohorts, and compared with conventional clinicopathological factors orCT evaluation. The predictive power of this miRNA signature in biopsysamples from University of Tokyo cohort was also validated (FIG. 10).

5. Statistical Analysis

Expression levels of each miRNA were shown as mean±standard error.Mann-Whitney U test was used to evaluate the statistical difference ofmiRNA expressions. Several clinicopathological characteristics werecompared between LNM positive and negative groups using the chi-squaretest or Fisher's exact test for categorical data. ROC curve and areaunder the curve (AUC) were used to evaluate the predictive value of eachmiRNA and miRNA signature for LNM. Prediction values calculated bylogistic regression model using miRNA expression levels were transformedinto z-score and were shown as risk score. Statistical calculations wereperformed using JMP Pro 11 statistical software (SAS Institute Japan,Tokyo, Japan), Medcalc version 16.1. (MedCalc Software, Belgium) andGraphPad Prism 7 (GraphPad Software Inc, CA, USA).

B. Results

1. The Discovery Phase in TCGA Database Identified Ten miRNAs to PredictLNM in T1 and 2 Colorectal Cancer

Logistic regression model with ROC back step elimination methodidentified 10 miRNA signature that best predicts LNM in T1-2 CRC in TCGAdatabase; mir-32, mir-181b (-1 and -2), mir-188, mir-193b, mir-195,mir-424, mir-425, mir-592, mir-3677, and mir-4326.

The expression of each miRNA between LNM positive and negative and ROCcurves are shown in FIG. 8. All of the miRNAs were highly upregulated inLNM positive samples compared to negative samples. Although the AUCvalue of each miRNA individually was insufficient to predict LNM, themiRNA signature which was constructed with these 10 miRNAs showed robustAUC value of 0.839 [95% CI(confidence interval): p=] to predict LNM inT1-2 CRC.

2. In Silico Validation of miRNA Signature Confirmed Good Performance ofthis Model

The inventors applied this model to T1 CRC patients of TCGA databaseincluding 2 LNM positive and 17 negative patients, and confirmed thatthis model performed well in T1 CRC patients with AUC value of 1.000(95% CI: p=) (FIG. 8).

Then, the inventors applied this model to GSE36650, which containedmiRNA expression data of Affymetrix microarray from 46 primary CRC siteand 43 LNM site. Because this dataset was generated by microarray,miR-3677 and miR-4326 were not included. Therefore, a miRNA signaturewas constructed using the other 8 miRNAs. This 8 miRNA signaturepredicted LNM site with AUC of 0.791 (95% CI: p=), and this resultsupported the idea that this miRNA signature was associated with LNM inCRC (FIG. 8).

3. Validation in Surgically Resected Samples from Kumamoto Univ. CohortShowed this miRNA Signature Performed Well in Clinical Samples

Kumamoto Univ. cohort included 7 LNM positive and 53 LNM negativepatients. The presence of lymphatic invasion was significantly morefrequent in LNM positive samples than that of negative samples(p<0.001). The other clinicopathological features such as tumor depth,poorly differentiated histology, and venous invasion were not associatedwith LNM (p=, 0.117, and 0.338, respectively) (Table 1).

TABLE 1 Associations between LN metastasis and clinicopathologicalfeatures Kumamoto Univ. Univ. Tokyo (N = 60) N (%) (N = 128) N (%) LNnegative LN positive LN negative LN positive Characteristics (N = 53) (N= 7) P value (N = 108) (N = 20) P value Gender 1.000 0.226 Male 34 (64)2 (29) 65 (60) 9 (45) Female 19 (36) 5 (71) 43 (40) 11 (55) Age (Years)0.117 1.000 <65 20 (38) 5 (71) 48 (44) 9 (45) ≥65 33 (62) 2 (29) 60 (56)11 (55) Tumor location 0.182 0.608 Colon 39 (74) 3 (43) 73 (68) 12 (50)Rectum 14 (26) 4 (57) 35 (32) 8 (40) Tumor depth (pm) 0.007 <1000 20 ( )0 (0) ≥1000 82 ( ) 18 ( ) Unavailable 6 ( ) 2 ( ) Tumor size (mm) 1.0000.647 <20 24 3 60 (56) 10 (50) ≥20 24 2 48 (44) 10 (50) Unavailable  5 20 (0) 0 (0) Tumor type Flat/Polypoid Depressed Histology 0.117 0.579(Differentiation) Well/Moderate  53 (100) 6 (86) 104 (96) 19 (95) Poor 0(0) 1 (14) 4 (4) 1 (5) Lymphatic invasion <0.001 <0.001 Negative 51 (96)0 (0) 99 (92) 10 (50) Positive 2 (4) 7 (100) 9 (8) 10 (50) Venousinvasion 0.338 0.224 Negative 42 (79) 4 (57) 61 (56) 8 (40) Positive 11(21) 3 (43) 47 (44) 12 (60) Risk factors 0.299 0.013 Negative  7 0 (0)16 ( ) 0 (0) Positive 19 7 (100) 87 ( ) 20 (100) Unavailable 27 0 (0) 5( ) 0 (0) Preoperative CEA 48 (91) 7 (100) 1.000 89 ( ) 19 (95) 0.302(ng/ml) <5 5 (9) 0 (0) 17 ( ) 1 (5) ≥5 0 (0) 0 (0) 2 ( 0 (0) unavailable

The expression levels of each miRNA between LNM positive and negativepatients are shown in FIG. 10. Consistent with the in silico analysis,the miRNA signature predicted LNM with AUC value of 0.908 (95% CI, p=),although most of the miRNAs were not statistically upregulated in LNMpositive samples compared with negative samples (FIG. 9A). When combinedwith the conventional risk factors (lymphatic invasion, venous invasion,and poorly histology) using logistic regression model, this model showedbetter AUC value of 0.992 to predict LNM in T1 CRC (FIG. 9A).

4. Second Validation in Large Number of Clinical Samples from Univ.Tokyo Cohort Showed Robustness of this miRNA Signature

Because Kumamoto Univ. cohort were relatively small cohort and almostall patients were classified as high risk by conventional pathologicalfeatures, the inventors validated the miRNA signature in another biggercohort which included 12.5% of risk negative and 83.6% of positivepatients (3.9% were not available).

Univ. Tokyo cohort included 20 LNM positive and 108 negative patients.Tumor depth was significantly deeper and the presence of lymphaticinvasion was significantly more frequent in LNM positive samples thanthat of negative samples (p=0.007 and <0.001, respectively). The otherclinicopathological features such as poorly differentiated histology andvenous invasion were not associated with LNM (p=0.579 and 0.224,respectively) (Table 1).

The expression levels of each miRNA between LNM positive and negativepatients are shown in FIG. 11. Consistent with the in silico analysisand that of Kumamoto Univ. cohort, the miRNA signature predicted LNMwith AUC value of 0.842 (95% CI, p=) (FIG. 9B) in this large cohort aswell. When combined with the conventional risk factors (tumor depth,lymphatic invasion, venous invasion, and poorly histology) usinglogistic regression model, this model showed better AUC value of 0.907to predict LNM in T1 CRC (FIG. 9B).

5. The Combination Model of 10 miRNA Signature with Risk FactorsImproved the Prediction Power of LNM in T1 CRC

The inventors further constructed LNM risk model with miRNA signature,conventional risk factors, and CT diagnosis using logistic regressionmodel. The combination model of miRNA signature, lymphatic invasion, andCT diagnosis better predicted LNM with AUC of 0.923 (95% CI, p=) (FIG.9C).

C. Discussion

This example demonstrations the construction of a robust miRNA signatureto predict LNM in T1 CRC using comprehensive method and validated in 19TCGA database samples and 188 of their own clinical specimens. Thissignature predicted LNM well with AUC of (sensitivity: and specificity:respectively).

One strength of the study described in this example was the comparisonwith conventional clinicopathological factors, which are the generalmethod to predict LNM. The prediction values of conventionalclinicopathological features were not sufficient, with the highest AUCbeing 0.70 of lymphovascular invasion (sensitivity: specificity:,respectively). This result is consistent with previous reports andhighlights the deficiencies in the current methods for predicting highrisk individuals. The inventors also evaluated the prediction power ofpreoperative CEA level, tumor budding, and CT, which are also generallyevaluated in clinical practices. However, these were also insufficient,when compared to the miRNA signature described in this example. Bycombining miRNA signature with these clinicopathological factors usinglogistic regression model, the predictive power of the model increasedup to AUC of 0.923 (sensitivity: specificity:).

Additionally, this example demonstrates, for the first time, theprediction power of the miRNA signature in the biopsy samples.Predicting LNM in biopsy samples would allow one to omit endoscopictreatment for the patients with high risk for LNM stratified by themiRNA signature, and this may eventually reduce the risk of endoscopicresection such as perforation or bleeding, physician's burden, andmedical cost.

Example 4—Identification of a Novel Network of miRNAs that RegulateStemness in Colorectal Cancer

Accumulating evidence suggests that a subset of cancer cells also knownas the “cancer stem cells” (CSCs) influence various clinical outcomes incancer, including tumor recurrence, metastasis and resistance tochemotherapy. Recently stemness has been recognized as a dynamic stategoverned by epigenetic modifiers including miRNAs. Despiteidentification of several self-renewal associated miRNAs, theirexpression profiles in CSCs remain unclear. In this example, theinventors systematically characterized miRNA expression patterns in CSCswith high vs. low CD44v6 expression through RNA-Seq. Subsequently, theinventors investigated the clinical significance of a novel miRNAidentified from this systematic discovery approach.

Colorectal CSCs from HCT116 and HT29 cells were grown asspheroid-derived cancer stem cells (SDCSCs). CD44v6⁺ and CD44v6⁻ CSCswere subdivided by FACS and characterized by small RNA-Seq.Differentially expressed miRNAs were subsequently confirmed in CD44v6⁺CSCs and chemoresistant cells. The expression of one such candidate,miR-1246, was assessed in a clinical patient cohort (n=144) by qRT-PCR.

MiRNA profiling identified a unique overall pattern of CD44v6⁺ SDCSCsindicative of high self-renewal capacity. It was noted that a panel ofestablished self-renewal suppressive-miRNAs were downregulated(including miR-34a, 101 and 200 family) in CD44v6⁺ CSCs, and discoveredupregulation of previously unreported miRNAs (miR-1246, 3605, 3182 and4284). KEGG pathway analysis indicated that these miRNAs regulateAkt-MAPK and Wnt signaling pathways. Subsequently, the inventorsselected miR-1246 and validated its expression in CD44v6⁺ SDCSCs andchemoresistant cells. Clinically, the expression of miR-1246 wassignificantly elevated in CRC tissues compared to corresponding normalmucosa, and this occurred in a stage-dependent manner in primary CRCs(FIG. 14). Furthermore, the expression of CD44v6 positively correlatedwith miR-1246 in CRC tissues (FIG. 15C). High miR-1246 expressionresulted in poor overall and disease free survival (FIG. 15A-B).

Using a systematic and comprehensive approach, the inventors haveidentified a unique network of dysregulated miRNAs in CD44v6 CSCsindicative of high degree of stemness features in cancer. In particular,miR-1246 was identified to be frequently over-expressed in CSCs as wellas chemoresistant cells and its expression was associated with poorprognosis in CRC patients. Collectively, the inventors have identified aunique group of previously unreported miRNAs which appear to haveimportant mechanistic roles in CSCs and could serve as a promisingpredictive biomarkers for recurrence and prognosis in patients with CRC.

Example 5—Genome-Wide Analysis Revealed a Robust Gene ExpressionSignature to Identify Lymph Node Metastasis in Submucosal ColorectalCancer

Due to recent advances in colonoscopic techniques, submucosal colorectalcancers (T1 CRCs) can now be removed endoscopically. Among these, 70% ofT1 CRCs are considered as “high risk” because they demonstrate presenceof lymphovascular invasion, poor differentiation, and the depth of tumoris >1000 um. However, post-surgical pathology results suggest that only˜10-15% of all T1 CRCs are truly lymph node positive, while majority ofhigh risk patients undergo unnecessary surgical treatments with currentcriteria. Since current pathological criteria have limitations,availability of molecular biomarkers that can identify ‘genuine highrisk patients with lymph node (LN) metastasis’ will reduce the burden ofsurgical overtreatment. Since gene expression-based classification ofCRC could identify patients with poor prognosis, the inventors sought toidentify a gene expression signature which can detect T1 CRCs with LNmetastasis.

Two independent publicly available genome-wide mRNA expression datasetswere used for mRNA biomarker discovery (n=125) and in-silico validation(n=56). Genome-wide unbiased gene expression signature was developedfrom The Cancer Genome Atlas (TCGA) RNA-Seq data by comparing theexpression profiles between 16 LN-positive and 109 LN-negative T½ CRCpatients. In addition to the selection of most differentially expressedgenes between the two groups, the inventors used (ROC) based back-stepelimination methodology to identify a robust mRNA panel. The gene panelwas validated in an independent publicly available dataset (n=56),followed by analytical validation in two independent T1 CRC patientcohorts (n=134 and n=67) using RT-PCR assays.

The in silico genome-wide comprehensive discovery led to theidentification of an eight gene mRNA classifier that significantlypredicted LN-metastasis with an AUC of 1.0, and the subsequentvalidation in an independent public data set resulted in an AUC of 0.93.The 8 genes include: AMT2 over-expression, MMP9 under-expression, FOXA1under-expression, C2CD4A under-expression, RCC1 under-expression, LYZunder-expression, MMP1 over-expression, and PIGR under-expression. TheRT-PCR based training and validation of this eight gene classifier intwo independent clinical cohorts robustly identified LNmetastasis-positive T1 CRC patients with an AUC of 0.90 (FIG. 16) and0.89 (FIG. 19), respectively.

A similar analysis was done for a 12 gene classifier that included AMT2over-expression, MMP9 under-expression, DEFA6 under-expression, FOXA1under-expression, MGAT5 under-expression, C2CD4A under-expression, RCC1under-expression, LYZ under-expression, MMP1 over-expression, NOS2under-expression, PIGR under-expression, CYP2B6 over-expression. Thevalidation was done in three separate cohorts (Tokyo, Kumamoto, andTMDU). The AUC values for these three cohorts were 0.9, 0.896, and 1.0,respectively. This analysis is shown in FIGS. 16, 19, and 22.

A similar analysis was done for a 16 gene classifier that included AMT,C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1, LYZ, MGAT5, MMP1, MMP9, NOS2, PIGR,PRAC1, RPL39L, RCC1, and SPAG16. This analysis is shown in FIGS. 25-27.

A similar analysis was done for a 15 gene classifier that included AMT,C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1, LYZ, MGAT5, MMP1, MMP9, NOS2, PIGR,PRAC1, RPL39L, and SPAG16. This analysis is shown in FIGS. 26-30.

A similar analysis was done for a 9 gene classifier that includedC2CD4A, DEFA6, MGAT5, MMP9, SPAG16, FOXA1, AMT, PRAC1, and RCC1. Thisanalysis is shown in FIGS. 31-32.

In conclusion, the inventors have identified a novel mRNA-basedclassifier that can detect high risk T1 CRCs with Lymph node metastasis.Further validation of these biomarkers in endoscopically collectedbiopsies will aid in clinical decision making and improving the clinicalmanagement of such patients.

Example 6—CCAT1 and CCAT2 Long Noncoding RNAs, Located within the8q.24.21 ‘Gene Desert’, Serve as Important Prognostic Biomarkers inColorectal Cancer

The 8q24.21 region is often referred to as a ‘gene desert’ due to lackof any important protein-coding genes, highlighting the potential roleof non-coding RNAs, including long non-coding RNAs (lncRNAs) locatedaround the proto-oncogene MYC. In this Example, the inventors havefirstly evaluated the clinical significance of altered expression oflncRNAs mapped to this genomic locus in CRC.

A total of 300 tissues, including 280 CRC and 20 adjacent normal mucosaspecimens were evaluated for the expression of 12 lncRNAs using qRT-PCRassays. The associations between lncRNA expression and variousclinicopathological features, as well as with recurrence free survival(RFS) and overall survival (OS) were analyzed in two independentcohorts.

The expression of CCAT1, CCAT1-L, CCAT2, PVT1, and CASC19 were elevatedin cancer tissues (P=0.039, <0.001, 0.018, <0.001, 0.002, respectively).Among these, high expression of CCAT1 and CCAT2 was significantlyassociated with poor RFS (P=0.049 and 0.022, respectively) and OS(P=0.028 and 0.015, respectively). These results were validated in anindependent patient cohort, in which combined expression of CCAT1 andCCAT2 expression was significantly associated with a poor RFS (HR:2.60,95% confidence interval [CI]: 1.04-6.06, P=0.042) and a poor OS(HR:8.38, 95% CI: 2.68-37.0, P<0.001). The inventors established a RFSprediction model which revealed that combined expression of CCAT1, CCAT2and carcinoembryonic antigen (CEA) was a significant determinant forefficiently predicting RFS in stage II (P=0.034) and stage III (P=0.001)CRC patients.

Several lncRNAs located in 8q24.21 locus are highly over-expressed inCRC. High expression of CCAT1 and CCAT2 significantly associates withpoor RFS and OS. The expression of these two lncRNAs independently, orin combination, serves as important prognostic biomarkers in colorectalcancer.

A. Materials and Methods

1. Patients and Sample Collection

This study included analysis of a total of 300 fresh frozen tissuespecimens, which encompassed 280 samples of primary colorectaladenocarcinoma and 20 matched corresponding normal mucosa tissues,collected from three institutes (Cohort 1; Mie University, Cohort 2;National Cancer Center Hospital, and Cohort 3; Tokyo Medical and DentalUniversity). Patients who underwent resection of their primary tumor andwere histologically confirmed to have a stage 0-IV CRC were included inthis study. Details of the clinicopathological features of the patientsinvolved in this study are shown in Table 1, and available at Annals ofOncology online. The flow chart for testing these 300 samples is shownin FIG. 35A.

SUPPLEMENTARY TABLE 1 Clinicopathological features of patients in thisstudy Cohort 1 (Mie Cohort 2 (NCC Cohort 3 (TMD Univ.) Hospital) Univ.)N = 20 N = 125 N = 135 Characteristics N (%) N (%) N (%) Gender Male 5(33) 70 (56) 77 (57) Female 15 (67) 55 (44) 58 (43) Age (Years) <75 13(65) — 99 (73) ≥75 7 (35) — 36 (27) Tumor Location Right sided — 23 (18)36 (27) colon Left sided — 42 (34) 44 (33) colon Rectum — 60 (48) 55(41) Tumor Size (mm) <50 — 78 (62) 64 (47) ≥50 — 47 (38) 65 (48)Unavailable 0 (0) 6 (4) Tumor Depth T1-2 — 27 (22) 28 (21) T3-4 — 98(78) 107 (79) Histology (Differentiation) Well/Moderate — 120 (96) 126(93) Poor — 5 (4) 9 (7) Lymphatic Invasion Negative — 80 (64) 59 (44)Positive — 45 (36) 76 (56) Venous Invasion Negative — 47 (38) 19 (14)Positive — 78 (62) 116 (86) Lymph Node Metastasis Negative — 56 (45) 77(57) Positive — 69 (55) 58 (43) Tumor Stage 0 3 (15) 0 (0) 0 (0) I 0 (0)19 (15) 22 (16) II 5 (25) 34 (27) 49 (36) III 7 (35) 55 (44) 34 (25) IV5 (25) 17 (14) 30 (22) Preoperative Serum CEA (ng/mL) <5 5 (33) 77 (62)76 (56) ≥5 15 (67) 48 (38) 59 (44) Median follow up period (Months) — 6960

2. Expression of lncRNAs Using Real-Time Quantitative ReverseTranscriptase Polymerase Chain Reaction

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)was performed using SYBR Green (Thermo Fisher Scientific, Waltham,Mass.). The results were normalized to the expression levels of ACTB.The sequences of the primers used in this study are listed in Table 2.

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)was performed using the QuantStudio™ 6 Flex Real Time PCR System(Applied Biosystems®, Foster City, Calif.) and expression levels wereevaluated using the QuantStudio™ 6 Flex Real Time PCR System Software.The relative abundance of target transcripts was evaluated utilizing 5ng of cDNA and SYBR Green (Thermo Fisher Scientific, Waltham, Mass.),and the results were normalized to the expression levels of ACTB usingthe 2^(−Δ) ^(Ct) method; ΔCt is the difference of Ct values between thelncRNA of interest and the normalizer. qRT-PCR was performed induplicate for each sample and the mean value was used to calculate theexpression levels. Normalized values were further log transformed andstandardized.

Supplementary TABLE 2 Primer sequences Forward Reverse CCAT1AGAAACACTATCACCTACGC CTTAACAGGGCATTGCTAAT CT CCAT1- CCACGTGCACATATTTGAATTGCATTCCCTGCTTAATACT L TG CA CCAT2 CCCTGGTCAAATTGCTTAACTTATTCGTCCCTCTGTTTTA CT TGGAT PVT1 TTGGGTCTCCCTATGGAATGGGAGAAGGCTCCAGGGAGTA PCAT1 TGAGAAGAGAAATCTATTGG GGTTTGTCTCCGCTGCTTTAAACC PCAT2 CTTAAGGCACTGATGCTCTC GTGCTGATGCCTCTGGAAAT A CASC8TCCAGCTTTGTGCTGATGAA CTTGCAACGTCAGTCCAAAA CASC11 CCCCAACACCTTCTTTGAACCGTCCAGTTGCTTTCCATC CASC19 ATTGGAGTGCCTGGGTTAGA TTTGGACAGCACCTTGAATGCASC21 CCAGAGGAGCCAAGAGAAGA CCAATGCTGTCCCACTCTGT PRNCR1CCAGATTCCAAGGG CTGA GATGTTTGGAGGCATCTGGT TA CCDC26 GGTGATGTGGTGCATCTGAGGCAACAACGGGAACTCTGAT MYC CGTCTCCACACATCAGCACA TCTTGGCAGCAGGATAGTCC A TTACTB AGAGCTACGAGCTGCCTGAC AGCACTGTGTTGGCGTACAG

3. Statistical Analysis

Receiver operating characteristic (ROC) curves with Youden's Indexcorrection were established to determine optimal cut-off values for eachlncRNA as it related to recurrence-free survival (RFS) and overallsurvival (OS). In multivariate analyses, a Cox proportional hazard modelwas used to identify clinical factors with a statistically significantinfluence on survival. Differences with a P value of <0.05 wereconsidered statistically significant. The inventors followed thecriteria of REporting recommendations for tumor MARKer prognosticstudies (REMARK) (See, for example, McShane L M et al., Nat Clin PractOncol 2005; 2: 416-422).

Expression levels of each lncRNA were shown as mean±standard error andwere compared using the paired Mann-Whitney test. Receiver operatingcharacteristic (ROC) curves with Youden's Index correction wereestablished to determine optimal cut-off value for each lncRNA as itrelated to recurrence-free survival (RFS) and OS. Patients were dividedinto two groups, high and low expression groups, by means of theYouden's index related to OS, and several clinicopathologicalcharacteristics were compared between the two groups using either thechi-square test or Fisher's exact test for categorical data or theMann-Whitney test for continuous variables. Pearson's coefficient wascalculated for correlation analysis. A RFS and OS curve were establishedfrom the time of resection using the Kaplan-Meier method and differenceswere evaluated using the log-rank test. Multivariate analyses wereperformed including all variables with a P value of <0.05 in univariateanalysis. In multivariate analyses, a Cox proportional hazard model wasused to identify factors with a statistically significant influence onsurvival. Differences with a P value of <0.05 were consideredstatistically significant. RFS prediction models were established usinga Cox-proportional hazard model using expression levels of lncRNAs andclinicopathological factors. ROC curves were established and area underthe curves (AUC) were compared between the each models. All statisticalcalculations were performed using JMP Pro 11 statistical software (SASInstitute Japan, Tokyo, Japan) and Medcalc version 16.1. (MedCalcSoftware, Belgium).

4. Total RNA Extraction and cDNA Synthesis

Total RNA was extracted from fresh frozen specimens using the RNeasyMini kit (QIAGEN) according to the manufacturer's instructions. Then,cDNA was synthesized from 2 μg of total RNA using the High Capacity cDNAReverse Transcription Kit according to the manufacturer'srecommendations (Thermo Fisher Scientific, Waltham, Mass.).

B. Results

1. The Screening Phase Identified Upregulation of Specific lncRNAs inColorectal Cancer

Twelve lncRNAs mapped to the 8q24.21 locus, which possess a HUGO GeneNomenclature Committee (HGNC) symbol, and have previously been suggestedto associate with cancer progression, were selected as candidates forinitial screening (FIG. 35B). The inventors compared the expressionlevel of each of the twelve lncRNAs in a Cohort 1, comprising of 20matched CRCs and normal mucosa (FIG. 36). Five of the twelve screenedlncRNAs; CCAT1, CCAT1-L, CCAT2, pvt1 oncogene (PVT1), and cancersusceptibility candidate 19 (CASC19), were significantly up-regulated incancer vs. normal tissues (P=0.037, <0.001, 0.017, <0.001, 0.002,respectively). The prostate cancer associated transcript 1 (PCAT1) wasat or below the limit of detection in most patients, and no significantdifferences were observed for the other six lncRNAs (P>0.05). Based onthese results, the inventors selected the five significantlyup-regulated lncRNAs (CCAT1, CCAT1-L, CCAT2, PVT1, and CASC19) forfurther evaluation.

2. The Testing Phase Revealed that High Expression of CCAT1, CCAT2 andPVT1 Associated with Poor Recurrence Free Survival and Overall Survivalin CRC Patients

Next, during the testing phase, the inventors examined the expression ofCCAT1, CCAT1-L, CCAT2, PVT1, and CASC19 in 125 CRC tissue specimens fromCohort 2. CCAT1-L and CASC19 could not be detected in 2 samples, andPVT1 could not in 3 samples. The expression levels of these five lncRNAswere analyzed in the context of various clinicopathologicalcharacteristics and prognosis of the patients. The detailed associationsbetween clinicopathological characteristics and expression of eachlncRNA are shown in Table 3.

TABLE 3 The associations between clinicopathological features and lncRNAexpression in NCCH cohort CCAT1 CCAT2 CCAT1-L Low High Low High Low HighN = 70 N = 55 P- N = 105 N = 20 P- N = 105 N=18 P- Characteristics (%)(%) Value (%) (%) Value (%) (%) Value Gender 0.126 0.557 0.798* Male 35(50) 35 (64) 60 (57) 10 (50) 47 (45) 7 (39) Female 35 (50) 20 (36) 45(43) 10 (50) 58 (55) 11 (61) Tumor Location 0.614 0.811* 0.611* Colon 35(50) 30 (55) 54 (51) 11 (55) 56 (53) 8 (44) Rectum 35 (50) 25 (45) 51(49) 9 (45) 49 (47) 10 (56) Maximum Tumor Size 0.800 0.615* 1.000* (mm)<50 43 (61) 35 (64) 64 (61) 14 (70) 100 (95)  18 (100) ≥50 27 (39) 20(36) 41 (39) 6 (30) 5 (5) 0 (0) Tumor Depth 0.699 0.568* <0.001 T1-2 16(23) 11 (20) 24 (23) 3 (15) 16 (15) 11 (61) T3-4 54 (77) 44 (80) 81 (77)17 (85) 89 (85) 7 (39) Histology 0.653* 0.588* 1.000* (Differentiation)Well/Moderate 68 (97) 52 (95) 101 (96)  19 (95) 66 (63) 11 (61) Poor 2(3) 3 (5) 4 (4) 1 (5) 39 (37) 7 (39) Lymphatic Invasion 0.940 0.447*0.288* Negative 45 (64) 35 (64) 69 (66) 11 (55) 65 (62) 14 (78) Positive25 (36) 20 (36) 36 (34) 9 (45) 40 (38) 4 (22) Venous Invasion 0.0800.314* 0.034* Negative 31 (44) 16 (29) 42 (40) 5 (25) 35 (33) 11 (61)Positive 39 (56) 39 (71) 63 (60) 15 (75) 70 (67) 7 (39) Lymph NodeMetastasis 0.016 0.220* 0.799 Negative 38 (54) 18 (33) 50 (48) 6 (30) 47(45) 9 (50) Positive 32 (46) 37 (67) 55 (52) 14 (70) 58 (55) 9 (50)Distant Metastasis 0.443* 0.148* 1.000* Absent 62 (89) 46 (84) 93 (89)15 (75) 91 (87) 16 (89) Present  8 (11)  9 (16) 12 (11) 5 (25) 14 (13) 2(11) Stage 0.020 0.324 0.610 I-II 36 (51) 17 (31) 47 (45) 6 (30) 44 (42)9 (50) III-IV 34 (49) 38 (69) 58 (55) 14 (70) 61 (58) 9 (50)Preoperative Serum 0.745 0.617* 0.795 CEA (ng/ml) <5 44 (63) 33 (60) 66(63) 11 (55) 64 (61) 12 (67) ≥5 26 (37) 22 (40) 39 (37) 9 (45) 41 (39) 6(33) PVT1 CASC19 Low High Low High N = 110 N = 12 P- N = 77 N = 46 P-Characteristics (%) (%) Value (%) (%) Value Gender 0.546* 0.498 Male 50(45) 4 (33) 32 (42) 22 (48) Female 60 (55) 8 (67) 45 (58) 24 (52) TumorLocation 0.230* 0.727 Colon 59 (54) 4 (33) 41 (53) 23 (50) Rectum 51(46) 8 (67) 36 (47) 23 (50) Maximum Tumor Size 1.000* 0.759 (mm) <50 68(62) 8 (67) 49 (64) 28 (61) ≥50 42 (38) 4 (33) 28 (36) 18 (39) TumorDepth 0.135* 0.029 T1-2 22 (20) 5 (42) 12 (16) 15 (33) T3-4 88 (80) 7(58) 65 (84) 31 (67) Histology 1.000* 1.000* (Differentiation)Well/Moderate 106 (96)  12 (100) 74 (96) 44 (96) Poor 4 (4) 0 (0)  3 (4)2 (4) Lymphatic Invasion 0.537 0.324 Negative 70 (64) 9 (75) 52 (68) 27(59) Positive 40 (36) 3 (25) 25 (32) 19 (41) Venous Invasion 0.365*0.283 Negative 40 (36) 6 (50) 26 (34) 20 (43) Positive 70 (64) 6 (50) 51(66) 26 (57) Lymph Node Metastasis 1.000* 0.270 Negative 51 (46) 5 (42)38 (49) 18 (39) Positive 59 (54) 7 (58) 39 (51) 28 (61) DistantMetastasis 1.000* 1.000* Absent 95 (86) 11 (92)  67 (87) 40 (87) Present15 (14) 1 (8)  10 (13)  6 (13) Stage 1.000* 0.287 I-II 48 (44) 5 (42) 36(47) 17 (37) III-IV 62 (56) 7 (58) 41 (53) 29 (63) Preoperative Serum0.534* 0.871 CEA (ng/ml) <5 69 (63) 6 (50) 48 (62) 28 (61) ≥5 41 (37) 6(50) 29 (38) 18 (39) CEA: carcinoembryonic antigen; *Fisher's exact test

The inventors thereafter evaluated the prognostic significance of eachlncRNA using the Kaplan-Meier analysis. High levels of CCAT1 and CCAT2expression were significantly associated with poor RFS (P=0.049 and0.022, respectively), and poor OS (P=0.028 and 0.015, respectively)(FIGS. 33A & 33B). Besides, high levels of CCAT1-L expression wassignificantly associated with poor RFS (P=0.048). However, expressionalterations in PVT1 and CASC19 did not demonstrate a significantassociation with tumor recurrence (P=0.178 and P=0.087, respectively)and patient survival (P=0.113 and 0.290, respectively), as shown in FIG.37. Accordingly, CCAT1 and CCAT2 were selected as candidate lncRNAs forfurther validation and evaluation of their prognostic potential inanother independent patient cohort.

3. Prognostic Significance of CCAT1 and CCAT2 lncRNAs was Validated inan Independent Cohort of CRC Patients

To further confirm and validate the prognostic significance of the twocandidate lncRNAs, the inventors analyzed another, large, independentcohort of 135 CRC tissues (Cohort 3). The high and low categoricalexpression cut-off thresholds were determined using Youden's index. Theassociations between each lncRNA expression and clinicopathologicalfeatures are shown in Table 4. RFS data was not available for 1 patientwith stage III CRC and excluded from RFS analysis.

TABLE 4 The association between clinicopathological factors and lncRNAsin TMD Univ. cohort CCAT1 CCAT2 Low High Low High N = 30 N = 105 P- N =101 N = 34 P- Characteristics (%) (%) Value (%) (%) Value Gender 0.3790.061 Male 15 (50) 62 (59) 53 (53) 10 (29) Female 15 (50) 43 (41) 48(48) 24 (71) Age (Years) 0.357 0.197 <75 20 (67) 79 (75) 77 (76) 22 (65)≥75 10 (33) 26 (25) 24 (24) 12 (35) Tumor Location 0.114 0.730 Colon 14(47) 66 (63) 59 (58) 21 (62) Rectum 16 (53) 39 (37) 42 (42) 13 (38)Maximum Tumor Size 0.183 0.729 (mm) <50 17 (57) 47 (45) 48 (48) 16 (47)≥50 11 (37) 54 (51) 47 (47) 18 (53) Unavailable 2 (15) 4 (4) 6 (6) 0 (0)Tumor Depth 0.799* 0.339* T1-2 7 (23) 21 (20) 19 (19)  9 (26) T3-4 23(77) 84 (80) 82 (81) 25 (74) Histology 0.207* 0.830* (Differentiation)Well/Moderate 30 (100) 96 (91) 94 (93) 32 (94) Poor 0 (0) 9 (9) 7 (7) 2(6) Lymphatic Invasion 0.190 0.649 Negative 10 (33) 49 (47) 43 (43) 16(47) Positive 20 (67) 56 (53) 58 (57) 18 (53) Venous Invasion 0.5651.000* Negative 3 (10) 16 (15) 14 (14)  5 (15) Positive 27 (90) 89 (85)87 (86) 29 (85) Lymph Node Metastasis 0.643 0.808 Negative 16 (53) 61(58) 57 (56) 20 (59) Positive 14 (47) 44 (42) 44 (44) 14 (41) DistantMetastasis 0.024* 0.815 Absent 28 (93) 77 (73) 79 (78) 26 (76) Present 2(7) 28 (27) 22 (22)  8 (24) Stage 0.747 0.963 I-II 15 (50) 56 (53) 53(52) 18 (53) III-IV 15 (50) 49 (47) 48 (48) 16 (47) Preoperative SerumCEA 0.642 0.649 (ng/ml) <5 18 (60) 58 (55) 58 (57) 18 (53) ≥5 12 (40) 47(45) 43 (43) 16 (47) CEA: carcinoembryonic antigen *: Fisher's exacttest

Next, the inventors evaluated the association between expression of bothlncRNAs with RFS and OS. Consistent with the findings in Cohort 2, highlevels of CCAT1 and CCAT2 expression were associated significantly withpoor RFS (P<0.001 and 0.010, respectively) as well as poor OS (P=0.011and 0.025, respectively) as shown in FIGS. 33C-D.

4. CCAT1 and CCAT2 Expression was an Independent Predictor of Poor RFSand OS in CRC Patients

The inventors next performed univariate and multivariate analyses usingthe Cox proportional hazard model in the validation cohort. Theunivariate analysis revealed that the age (>75) (HR:2.27, 95%CI=1.05-4.74, P=0.037), the presence of lymph node metastasis (HR:2.89,95% CI=1.38-6.05, P=0.005), high pre-operative serum carcinoembryonicantigen (CEA) levels (HR:2.98, 95% CI:1.43-6.32, P=0.004), high CCAT1expression (HR:3.88, 95% CI: 1.67-8.39, P=0.003), and high CCAT2expression (HR:2.55, 95% CI: 1.19-5.31, P=0.017) were significantlyassociated with poor RFS, while other clinicopathological factors andhigh CCAT1 and CCAT2 expression also demonstrated a significantassociation with OS as well (CCAT1 expression: HR: 4.06, 95% CI:1.47-16.8, P=0.004, CCAT2 expression: HR: 2.04, 95% CI: 1.05-3.84,P=0.036, Table 5).

TABLE 5 Univariate analysis of RFS and OS using a Cox proportionalhazard model RFS OS Variables HR 95% CI P-value HR 95% CI P-value Gender1.06 0.50-2.20 0.881 1.18 0.62-2.19 0.611 Female/Male Age ≥75/<75 2.271.05-4.74 0.037 1.43 0.66-2.84 0.345 (Years) Tumor Location 1.750.84-3.71 0.131 0.94 0.49-1.75 0.837 Rectum/Colon Tumor Depth 2.500.97-8.51 0.059 2.24 0.96-6.54 0.062 T3-4/T1-2 Histology(Differentiation) 1.70 0.41-4.85 0.417 1.62 0.49-4.06 0.389Poor/Well-Moderate Lymphatic Invasion 1.20 0.58-2.55 0.614 1.800.95-3.60 0.073 Positive/Negative Vascular Invasion 2.06 0.72-8.64 0.1937.78 1.69-138 0.003 Positive/Negative Lymph Node Metastasis 2.891.38-6.05 0.005 4.69 2.41-9.82 <0.001 Positive/Negative Stage — — — 7.093.32-17.5 <0.001 III-IV/I-II Preoperative Serum 2.98 1.43-6.32 0.0043.78 1.96-7.72 <0.001 CEA ≥5/<5 (ng/mL) CCAT1 expression 3.88 1.67-8.390.003 4.06 1.47-16.8 0.004 High/Low CCAT2 expression 2.55 1.19-5.310.017 2.04 1.05-3.84 0.036 High/Low RFS: recurrence free survival, OS:overall survival, HR: hazard ration, CI: confidence interval, CEA:carcinoembryonic antigen.

Interestingly, multivariate analysis revealed that the expression levelsof CCAT1 (HR: 2.52, 95% CI: 1.07-5.56, P=0.036) and CCAT2 (HR: 2.39, 95%CI: 1.10-5.08, P=0.029) were independent factors for predicting poor RFSand poor OS (CCAT1: HR: 5.90, 95% CI: 2.09-24.7, P<0.001 and CCAT2: HR:2.40, 95% CI: 1.22-4.59, P=0.011; Table 1). Taken together, theinventors successfully validated the prognostic significance of bothCCAT1 and CCAT2 expression as important prognostic biomarkers inmultiple cohorts of CRC patients.

5. CCAT1 and CCAT2 Expression Significantly Correlated with MYC inColorectal Cancer

Since there have been suggestions that lncRNAs mapped to the 8q24.21locus may be associated with MYC, the inventors evaluated therelationship between expression of CCAT1 and CCAT2 with MYC. Theinventors evaluated MYC expression by qRT-PCR in the Cohort 3. BothCCAT1 and CCAT2 expression were significantly correlated with MYCexpression (r=0.66, P<0.001 and r=0.74, P<0.001, respectively; FIG. 38,further supporting the functional and clinical relevance of thesefindings in colorectal cancer.

6. Combined Expression of CCAT1 and CCAT2 is a Superior Predictor forRFS and OS in CRC Patients

Due to correlative functional nature of CCAT1 and CCAT2, it was soughtto examine associations for their combinatorial expression in predictingRFS and OS. In this regard, the inventors categorized all patients intothree groups; a) with elevated expression of both CCAT1 and CCAT2, b)with elevated expression of either CCAT1 or CCAT2, and c) with lowexpression of both CCAT1 and CCAT2. By performing such analysis, theinventors discovered that the patients that co-expressed high levels ofCCAT1 and CCAT2 correlated with poorer RFS compared to other groups(P=0.049 both high vs. either high, P<0.001 both high vs. both low,respectively; FIG. 34A). In the case of OS, the three groups were morespread out, such that both high vs. either high (P=0.038) and both highvs. both low (P=0.002) were significantly different from one another,and demonstrated that patients with high levels of both CCAT1 and CCAT2had the worst OS. Furthermore, multivariate analysis by combiningexpression levels of both CCAT1 and CCAT2 revealed that the group ofpatients with high co-expression of CCAT1 and CCAT2 had higher hazardratios for RFS (HR:2.60, 95% CI: 1.04-6.06, P=0.042) and also for OS(HR: 8.38, 95% CI: 2.68-37.0, P<0.001) compared with the both lowexpression group (Table 6).

TABLE 6 Multivariate analyses of RFS and OS using Cox proportionalhazard model CCAT1 CCAT2 CCAT1 + CCAT2 P- P- P- Variables HR 95% CIvalue HR 95% CI value HR 95% CI value Multivariate analysis for RFS Age≥75/<75 2.28 1.05-4.85 0.039 2.23 1.01-4.74 0.046 2.20 1.00-4.69 0.050(Years) Lymph Node 2.30 1.09-4.89 0.029 2.80 1.31-5.98 0.008 2.861.28-6.64 0.011 Metastasis Positive/Negative Preoperative 2.49 1.17-5.350.017 2.60 1.23-5.59 0.013 2.67 1.24-5.85 0.013 Serum CEA ≥5/<5 (ng/mL)LncRNA 2.52 1.07-5.56 0.036 2.39 1.10-5.08 0.029 2.60 1.04-6.06 0.042expression High/Low Multivariate analysis for OS Vascular Invasion 3.730.75-67.7 0.124 3.84 0.76-69.9 0.116 3.97 0.78-72.5 0.107Positive/Negative Lymph Node 0.65 0.25-2.22 0.447 0.66 0.25-2.26 0.4690.60 0.22-2.06 0.375 Metastasis Positive/Negative Stage 9.07 2.31-31.20.003 7.86 2.01-26.8 0.005 9.94 2.52-34.5 0.002 III-IV/I-II Preoperative2.26 1.15-4.73 0.017 2.25 1.14-4.71 0.019 2.27 1.15-4.77 0.017 Serum CEA≥5/<5 (ng/mL) LncRNA 5.90 2.09-24.7 <0.001 2.40 1.22-4.59 0.011 8.382.68-37.0 <0.001 expression High/Low RFS: recurrence free survival, OS:overall survival, HR: hazard ration, CI: confidence interval, CEA:carcinoembryonic antigen

7. A RFS Prediction Model Highlighted the Prognostic Potential of CCAT1and CCAT2 in Colorectal Cancer

The inventors constructed a RFS prediction model with variouscombinations of parameters including serum CEA and the expression levelsof CCAT1 and CCAT2 using the Cox proportional hazard model, in which thearea under the curves (AUCs) for each variable were compared byconstructing ROCs for 5 years' recurrence in 80 stage I-III CRC patients(53 non-recurrence with follow-up ≥5 years and 27 recurrence within 5years) (FIG. 34B). The combination of CCAT1, CCAT2 and CEA expressionyielded the greatest AUC of 0.793 (95% CI: 0.687-0.876). Thereafter, theinventors evaluated the RFS using this model in stage II and stage IIICRC patients separately. This model efficiently distinguished RFS inboth stage II and stage III CRC patients by employing the Kaplan-Meiercurve analysis (P=0.034 and 0.001, respectively, FIG. 34C).

C. Discussion

In the current example, the inventors, for the first time, haveperformed a comprehensive investigation on the clinical significance oflncRNAs mapped to the 8q.24.21 locus ‘gene desert’, in CRC. It was foundthat five of the twelve lncRNAs in this locus were upregulated in CRC,and among them, high expression of CCAT1 and CCAT2 significantlyassociated with poor RFS and OS in CRC patients, in two independentcohorts. This example provides first clinical validation to suggest thatCCAT1 and CCAT2 play an essential role in CRC progression, which may inpart me mediated through their interactions with MYC.

Following a potentially curative surgery, approximately 30% of CRCpatients will often eventually develop metastases, in spite of adjuvanttherapies, such as chemotherapy and radiochemotherapy. Although adjuvantchemotherapy provides significant survival benefit in stage IIIpatients, its clinical significance is stage II CRCs remainscontroversial, since 20-30% of these patients eventually experiencetumor relapse. Recently, it was suggested that a subset of stage II CRCpatients may benefit from adjuvant chemotherapy, but a prioriidentification of such patients remains presents a clinical challenge.In this example, the inventors have established a RFS prediction modelusing a Cox-proportional hazard analysis by utilizing the expressionlevels of CCAT1 and CCAT2 with serum CEA. It was demonstrated that thecombination of CCAT1, CCAT2 and CEA expression levels were the bestpredictors of RFS. In addition, the model predicted RFS not only instage III CRC patients, but in stage II CRC patients as well. Hence, theprognostic biomarkers identified in this study, and the novel RFSprediction model may serve as an actionable approach for clinicaldecision-making for adjuvant therapy in stage II CRC patients.

In conclusion, several lncRNAs located in 8q24.21 are highly expressedin CRC and may be associated with carcinogenesis or tumor progression.Among these over-expressed lncRNAs, the inventors identified that CCAT1and CCAT2 are associated with tumor recurrence and poor prognoses, andevaluating the expression of these two lncRNAs may provide useful,actionable, biomarkers for predicting tumor recurrence or prognosis inCRC patients.

Example 7—Plasma Levels of piRNAs as Biomarkers for Prognosis andPredicting Tumor Recurrence in Colorectal Cancer Patients

Accumulating evidence in recent years indicates that small non-codingRNAs (sncRNAs), such as miRNAs, lncRNAs, snoRNAs and piwi-interactingRNAs (piRNAs), play a central role in many diseases, including cancer.In this regard, although the mechanistic role piRNAs play in cancerpathogenesis continues to evolve, it is believed that they may functionjust like miRNAs in causing transcriptional repression of gene targetsor by inducing hypermethylation of specific tumor suppressor genes.Considering their small size and stability in biological fluids, andlack of any reports for their role as disease biomarkers in cancer, theinventors undertook this study to systematically and comprehensivelyevaluate the piRNA expression patterns and their biomarker potential intissue and plasma specimens from colorectal cancer (CRC) patients.

The inventors performed a discovery phase by performing statisticalanalysis on RNA-Seq data from TCGA, to identify differentially expressedpiRNAs between early (stage I/II) vs. late (stage IV) CRCs. Thereafterthe inventors evaluated the clinical significance of these piRNAs in 405surgically resected tissue and 145 plasma samples from two, independentpatient cohorts (testing: n=202, validation: n=203) by quantitative PCR,and analyzed the results with various clinicopathological features andpatient survival.

The inventors identified a panel of 3 piRNAs (piR61919, piR30652, andpiR31111), which were significantly up-regulated in cancer vs. matchednormal tissues in CRC patients. Kaplan-Meier curves revealed that highpiR61919 and piR30652 expression were significantly associated with pooroverall survival (p<0.001 and 0.016, respectively), and these resultswere subsequently validated in an independent validation cohort (p=0.016and 0.005, respectively). More importantly, high levels of piR61919 andpiR30652 in plasma specimens significantly associated with poorrecurrence free survival (p=0.029 and <0.001, respectively) in stageII-III CRC patients. A Cox regression model which included combinedexpression levels of piR61919 and piR30652 revealed a robust AUC valueof 0.779 to predict 5 years' recurrence in stage II-III CRC patients.

The inventors, for the first time, demonstrate that piR61919 andpiR30652 expression levels in tissue, but more intriguingly in plasma,may serve as novel and clinically useful biomarkers to predict prognosisand tumor recurrence in CRC.

Example 8: Gene-Specific 5hmC Levels as Biomarkers for DiseaseProgression and Survival in Colorectal Cancer

Aberrant DNA demethylation, constitutes an important epigeneticalteration in various diseases including cancer. Active demethylation ofmethylated cytosine (5mC) involves TET genes-mediated generation ofintermediates such as 5hmC, 5fC and 5caC (FIG. 39). Among theseintermediaries, 5hmC is relatively abundant in human genome and ispivotal in cellular functioning because of its association with DNArepair genes and other DNA binding proteins. Recent reports havesuggested global depletion of 5hmC levels in multiple cancers. However,since alterations in 5hmC content on a gene-specific level has importantfunctional consequences and potential clinical significance, theinventors undertook the present study to examine and compare global andgene-specific 5hmC levels in colorectal cancer (CRC) and theirprognostic implications.

Global 5hmC levels were measured in matched tumor and adjacent normalsamples in two CRC patient cohorts (n=25 and 100) by ELISA. The geneexpression data (shown as log fold change of 5hmC for the two cohortsare shown in FIGS. 40-42 and 46-47. Eight candidate genes were selectedfor gene-specific 5hmC level measurements based on publicly availabledatasets and their functional role in CRC pathogenesis (P2RX4, PTAFR,CRISPLD2, FKBP4, PDE4DIP, VHL, TGFBI and SFRP2). Gluc-MS-qPCR wasperformed for gene-specific 5hmC measurement. Wilcoxon signed rank testalong with survival analyses were performed, and TCGA dataset wasanalyzed for expression profiles of significantly altered genes.

Global 5hmC levels were significantly lower in cancer vs. normal tissuesin CRC patients (p<0.001). Stage-wise decrease in gene-specific 5hmclevels (trend test p values <0.001 for P2RX4, SFRP2, CRISPLD2 and FKBP4)was observed. While the inventors did not observe any prognostic role ofglobal 5hmc levels, lower 5hmC levels in P2RX4, CRISPLD2 and FKBP4 weresignificantly associated with poor overall survival (p=0.02, p=0.02 andp=0.006 respectively) (FIGS. 43 & 48). Furthermore multivariate Coxregression analysis showed FKBP4 to be an independent prognostic factorfor overall survival (p=0.008). Lower levels of gene-specific 5hmCcontent significantly correlated in patients with distant metastasis(CRISPLD2, p=0.008; FKBP4, p=0.004), while lower global 5hmC contenttogether with 5hmC levels of two candidate genes were associated withliver metastasis (Global 5hmC, p=0.04; CRISPLD2, p=0.009 and FKBP4,p=0.01) (FIG. 45). A large majority of lymph node positive samples(31/40) had significantly lower levels of P2RX5-specific 5hmC (p=0.02).TCGA dataset showed reduced expression of P2RX4, CRISPLD2 and SFRP2(p<0.001; p=0.008; p<0.001 respectively), which corroborated with thehypothesis.

ROC curves were conducted for the association of the biomarkers withpoor prognosis in colorectal cancer. The AUC values are shown below:

Biomarker AUC Global 5 hmC modification 0.75 P2RX4 5 hmC modification0.81 SFRP2 5 hmC modification 0.76 CRISPLD2 5 hmC modification 0.79FKBP4 5 hmC modification 0.66 Combination of global, P2RX4, CRISPLD2,and FKBP4 0.89 Combination of global, P2RX4, SFRP2, CRISPLD2, and FKBP40.92

Global 5hmC levels were significantly lower in CRC tissues compared tonormal specimen. In particular, gene-specific 5hmC levels emerged assuperior prognostic biomarkers in CRC patients, and may serve asimportant clinical tools for determining patient survival and improvingpatient management.

Example 9: ITGBL1 is a Novel Epithelial MesenchymalTransition-Associated Prognostic Biomarker in Colorectal Cancer

Colorectal cancer (CRC) ranks as the third leading cancer worldwide, andits incidence continues to rise gradually, highlighting the need tostratify the risk of recurrence after curative surgery. Recently,several genes have been identified which appear to associate withmetastasis, as they mediate epithelial-to-mesenchymal transition (EMT)in cancer. This study aimed to identify novel EMT and cancerrecurrence-associated biomarkers through systematic and comprehensivediscovery and validated strategy in multiple, independent CRC cohorts.

Two independent gene expression microarray datasets (n=173 and n=307respectively) were used to identify novel metastasis-recurrencebiomarkers for CRC. Following carefully selection and prioritization ofbiomarkers, the inventors selected a candidate gene and validated itsperformance as a recurrence marker in a large testing cohort (n=566),and two independent clinical validation cohorts (n=201, n=475,respectively). To confirm the protein expression of ITGBL1 in cancer,immunohistochemistry (IHC) was performed in paired 33 primary CRCs andadjacent normal mucosa, as well as a subset of liver and lung metastasestissues. In addition, we used Gene Set Enrichment Analysis (GSEA) todetermine the functional role of ITGBL1 in CRC.

During the discovery step, gene expression profiles from differentiallyexpressed genes between recurrence positive and negative primary CRCs,as well as evaluation of the metastatic sites compared with primary CRC,identified ITGBL1 as a most promising candidate biomarker. Highexpression of ITGBL1 associated with poor overall survival (OS) in stageI-IV patients and worse disease-free survival (DFS) in stage I-IIIpatients. Subgroup validation of these results in two large andindependent patient cohorts confirmed these findings and demonstratedthat high ITGBL1 expression correlated with shorter DFS in stage II andIII CRC patients. In addition, high ITGBL1 expression emerged as anindependent prognostic factor for DFS in stage II and III patients. IHCanalysis revealed that both early stage CRCs and adjacent normal colonicmucosa displayed low ITGBL1 expression, while ITGBL1 expressiongradually increased from tumor surface to the invasive front in latestage cancer, indicating that ITGBL1 may facilitate EMT process andpromote a more aggressive phenotype in CRC.

High expression of ITGBL1 in primary tumors was associated with tumorrecurrence in CRC patients after curative surgery. Collectively, we haveidentified ITGBL1 as a novel EMT-associated biomarker which could beused for risk stratification for metastatic potential in CRC.

Example 10: piRACC, a Novel Oncogenic piRNA, Promotes Tumor Progressionand Predicts Unfavorable Prognosis in Colorectal Cancer

Colorectal cancer (CRC) constitutes a major heath burden in most westerncountries. Very recent and relatively limited evidence indicates thatpiwi-interacting RNAs (piRNAs) play crucial roles in several types ofcancers. However, the biological involvement of piRNAs in colorectalcarcinogenesis remains elusive. In this study, the inventors performedsystematic piRNA expression profiling between CRC and paired normaltissues by small RNA-Seq, and identified piRNA DQ570994 (named as piRACCfor piRNA Associated with Colorectal Cancer), as a novel, differentiallyexpressed piRNA in CRC. piRACC was found to be frequently overexpressedin CRC tissues from multiple independent patient cohorts (with a5.49-7.0 fold increase and P<0.01 in each cohort). To interrogate theclinical significance of piRACC in CRC, the inventors evaluated itsexpression level in 771 CRC patients from a TCGA dataset, a clinicaltesting cohort and a validation cohort. The overexpression of piRACC wassignificantly associated with several known clinicopathological riskfactors (advanced T-stage, P=0.0008 and P=0.0434 in testing andvalidation cohort respectively; lymph node involvement, P=0.025 andP=0.0025 in testing and validation cohort respectively; and distantmetastasis, P=0.0319 and P=0.0027 in testing and validation cohortrespectively), and furthermore, patients with high expression of piRACChad a shorter overall survival compared to those with low expression ofpiRACC (HR=2.387, P=0.0026 in testing cohort; HR=3.208, P=0.0002 invalidation cohort). Multivariate Cox's regression analysis demonstratedthat high piRACC expression was an independent predictor for pooroverall survival in CRC (HR=1.965, P=0.0298 in testing cohort; HR:2.9347, P=0.0025 in validation cohort). The inventors supported thesefindings by performing a series of functional assays and demonstratedthat piRACC exerts its oncogenic function through promotion of cellsurvival, migration and invasion as well as suppression of apoptosis.Consistent with its aggressive biological and clinical phenotype, theinventor's microarray data revealed a subset of genes regulated bypiRACC are enriched in cancer-related pathways as indicated by IngenuityPathways Analysis (IPA). By using bioinformatics approaches, nine tumorsuppressor genes (ATF3, BTG1, DUSP5, FAS, NFKBIA, UPP1, SESN2, TP53INP1and MDX1) were predicted to be direct potential targets of piRACC due tosequence complementarity. The regulation of the expression of thesetarget genes by piRACC was subsequently validated in CRC cell lines andinversely associated with piRACC expression in CRC tissues. Inconclusion, this example, for the first time, has provided evidence fora novel piRNA (piRACC), which promotes CRC pathogenesis and may be animportant prognostic biomarker in CRC.

Colorectal cancer (CRC) constitutes a major public heath burden, beingthe third most commonly diagnosed cancer and the fourth cause of cancerdeath worldwide. Interestingly, recent reports showed the incidence ofcolorectal cancer in Asian countries, which were previously consideredas low rate, has increased dramatically in last two decades. Consideringthe mortality and burden of this disease, it is imperative toinvestigate the prevention and treatment strategies for the managementof this malignancy.

CRC develops as a consequence of genetic and epigenetic alterations,which occur with tumor initiation and progression. Due to the molecularheterogeneity, the prognosis and response to chemotherapy betweenindividual patients can vary largely. However, current guidelinesselected “risk” patients solely based on clinicopathological factors andintraoperative findings, suggesting potential risk for under orover-treatment for CRC patients. Therefore, readouts of disease biologyby novel molecular targets would be of great value in prognosisassessment and/or cancer treatment.

The goals of the study set forth in this example were to systematicallyand comprehensively interrogate the molecular contributions of the piRNAsuper family members in colorectal cancer, identify specific piRNAs thataberrantly expressed between tumor and normal tissues, and decipherwhether these candidate piRNAs may have translational relevance asclinically relevant disease biomarkers. In addition, to support theclinical findings, the functional and mechanistic role of piRNAs inhuman colorectal cancer will also be investigated.

A. Methods

1. Patients and Study Design

To identify CRC-associated piRNA, the inventors prepared small RNAsequencing libraries from 4 frozen cancer tissues and paired normalmucosa (NM) specimens, which were collected at the Mie University,Japan. To confirm the expression level of candidate piRNA between cancerand normal tissues, the inventors measured their expression levels inmatched cancer and normal frozen tissues from 3 different cohorts of MieUniversity, Japan (n=20), Shanghai Tenth People's Hospital, China (n=20)and Okayama University Medical Hospital, Japan (n=18). To investigatethe prognostic value of candidate piRNAs in CRC, the inventors analyzedpiRACC expression pattern in three different cohorts of a combined totalof 771 CRC patients from TCGA dataset, clinical testing cohort andvalidation cohort. The expression profile of piRNAs from TCGA database(n=387) was characterized by Martinez et al. (Sci Rep 5, 10423 (2015)).The inventors then analyzed candidate piRNAs expression in clinicaltesting cohort (n=195, Shanghai Tenth People's Hospital) and asubsequent validation cohort (n=189, Okayama University MedicalHospital). Both testing and validation cohort are FFPE samples.Micro-dissection was performed to enrich tumor cells from the FFPEsamples and the baseline characteristic of these patient cohorts isdescribed in Table 1. To further understand the mechanistic correlationof piRNA expression in CRC, the inventors determined its expressionpattern in the context of its target genes in fresh frozen samples(n=159). Written informed consent was obtained from all patients and thestudy was approved by the institutional review boards of allparticipating institutions. All CRC patients were followed up forsurvival for at least 5 years duration from their date of surgery.Patients treated with radiotherapy or chemotherapy before surgery wereexcluded from the study.

TABLE 1 Clinicopathological characteristic and piRACC expression intesting and validation cohort Testing cohort Validation cohort Cases LowHigh P^(c) Cases Low High P^(c) Gender Male 91 45 46 0.9391 110 52 580.4255 Female 104 52 52 79 42 37 Age ≤69^(a)/66^(b) 100 55 45 0.133 10045 55 0.1687 >69766^(b) 95 42 53 89 49 40 Tumor location Distal 150 8268 *0.0123 121 55 66 0.1174 Proximal 45 15 30 68 39 29 Histological typeWell/moderate 175 90 85 0.0566 180 90 90 0.7456 Poor 18 5 13 9 4 5Unknown 2 — — — — — Pathological T category pT1-3 48 34 14 **0.0008 15482 72 *0.0434 pT4 147 63 84 35 12 23 Lymph node metastasis Negative 13273 59 *0.025 85 53 32 **0.0025 Positive 63 24 39 100 40 60 Unknown — — —4 — — Distant metastasis Negative 187 96 91 *0.0319 143 80 63 **0.0027Positive 8 1 7 46 14 32 Stage I 29 21 8 **0.006 28 18 10 **0.0052 II 9951 48 53 33 20 III 59 24 35 62 29 33 IV 8 1 7 46 14 32 ^(a)The medianage of testing cohort is 69. ^(b)The median age of validation cohort is66. ^(c)Pearson chi-squared testing was used - compare the correlationbetween piRACC expression and clinical variables. *P < 0.05; **P < 0.01.

2. Small RNA-Seq Analysis

For RNA-seq, 1 μg of total RNA was used for library preparation withIllumina TruSeq small RNA sample preparation Kit. Linker sequences weretrimmed off from the 50 nucleotide raw sequences by using fastx_clipperwith at least 8 basepair match. All the trimmed sequences must be notshorter than 9 nucleotides long. Next, the trimmed sequences werefiltered by human miRNAs reported by mirBase. The remaining small RNAsthat could be mapped to human genome hg38 were matched to known piRNAscollected from piRNA bank (found on the web at pirnabank.ibab.ac.in) andpirbase (see for example, the world wide web atregulatoryrna.org/database/piRNA/) databases. DESeq was employed toidentify differentially expressed piRNAs in colorectal cancer patients(with ≥2 fold change and P-value≤0.01).

3. piRNA Quantification by qRT-PCR

Expression of identified piRNAs (DQ593356, DQ596309, DQ593752 andDQ570994) was analyzed using Custom TaqMan small RNA assays (AppliedBiosystems, Foster City, Calif., USA), and U6 expression was used as anendogenous control for data normalization, as described previously. Theaverage expression levels of tissue piRNAs were normalized against U6using the 2^(−ΔCt) method.

4. Gene Expression Analysis by Quantitative Reverse TranscriptionPolymerase Reaction (qRT-PCR)

The qRT-PCR assays were performed using QuantStudio 6 Flex Real-Time PCRSystem (Applied Biosystems Foster City, Calif.). 500 ng of total RNA wasconverted to cDNA using High-Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif.). Real-time PCR was thereafterperformed using Fast SYBR Green Master Mix (Applied Biosystems, FosterCity, Calif.). The relative expression of target genes was determined by2^(−Δct) method. GAPDH were used as normalizers. The primer sequencesused are shown in Table 2:

TABLE 2 Primers sequence Gene Forward (5′→3′) Reverse (5′→3′) MXD1GCTGAACATGGTTATGCCTCC AGCCCGTCTATTCTTCTCCAT TTC DUSP5TCCTGAGTGTTGCGTGGATG GGGCCACCCTGGTCATAAG BTG1 GGAGCTGCTGGCAGAACATTAGTGCTGCCTGTCCAATCAGA TP53INP1 CTCACGGGCACAGAAGTGGAA ATCCACTGGGAAGGGCGAAFAS GTACGGAGTTGGGGAAGCTC ACAGACGTAAGAACCAGAGGT SENS2TCGCTCTCCTCCTTCGTGTT TCAAAGCCCCCAGAGTTGTTC NFKBIA CCCCTACACCTTGCCTGTGCACGTGTGGCCATTGTAGTTG UPP1 GAGTGGGCTTGGTGAGGTG CAGGACCCGTCAGAGGAGAG ATF3GCCGAAACAAGAAGAAGGAGA TCGTTCTTGAGCTCCTCAATC GAPDH TGTAGTTGAGGTCAATGAAGACATCGCTCAGACACCATG GG

5. Cell Lines, RNA Oligos, Antisense and Transfection

HCT116 and SW480 were obtained from the American Type Culture Collection(ATCC, Rockville, Md.) and cultured in Iscove's modified Dulbecco'smedium (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovineserum (FBS) and antibiotics (100 U/ml penicillin and 100 μg/mlstreptomycin) at 37° C. in 5% humidified CO2 atmosphere. These celllines were periodically authenticated using a panel of genetic andepigenetic biomarkers.

For the overexpression of piRACC in CRC cell lines, HCT116 and SW480were transfected in biological triplicate with either a single-strandedRNA oligos (5′AGC CCU GAU GAU GCC CAC UCC UGA GC-3′ with 2′-O-methylated3′-end) or a single-stranded scrambled RNA control (5′-UCA CAA CCU CCUAGA AAG AGU AGA-3′ with 2′-O-methylated 3′-end). For the inhibition ofpiRACC in CRC cell lines, the antisense were designed as describedpreviously. The 2′-O-Me-modified antisense sequence was 5′-CUUA GCT CAGGAG TGG GCA TCA TCA GGG CT ACCUU-3′, while negative scrambled controlwas 5′-CUUA TC aGG ACT gCT ACt GGT GcG GAC gCG ACCUU-3′.

For the transfections, colorectal cancer cells were transfected with RNAoligos or antisense at a final concentration of 100 nmol/L usingLipofectamine RNAi MAX(Invitrogen) and Opti-MEM (Gibco) according to themanufacturer's instructions.

6. MTT and Colony Formation Assay

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay(Sigma, St. Louis, Mo., USA) was used to evaluate cell proliferation.Cells were seeded at 1×10³ cells per well in 96 well plates. MTT wasadded at 0h-, 24-, 48h-, 72h-hour time-points. Absorbance was read at570 nm using Infinite 200 Pro multi-reader (Tecan Group Ltd,Morrisville, N.C.). For colony formation assay, 500 cells were seeded ineach well of 6 well plates and incubated for 10 days. The colonies werestained with crystal violet and counted.

7. Cell Invasion, Migration and Apoptosis Assay

Migration and invasion assays were performed using Boyden chambers(Corning, Corning, N.Y.) using 8 μm-size pore membrane coated withmatrigel (for invasion assays) or without matrigel (for migrationassays). Transfected cells in serum-free medium were seeded onto eachinsert at a density of 2×105 cells/insert, with culture mediumcontaining 10% FBS in the bottom well. Following 24h incubation,non-invading cells were removed by scraping the top of the membrane.Invaded cells on the bottom of the membrane were then fixed and stainedusing diff-quick staining kit (Thermo Scientific, Rockford, Ill.).Stained cells were counted using a light microscope. For apoptosisassays, Muse Annexin V and dead cell kit (Millipore, Billerica, Mass.)were used according to the manufacturer's instructions.

8. Immunofluorescence (IF)

For IF, cells were fixed by 4% paraformaldehyde for 15 min, washed withPBS and blocking buffer (3% FBS, 1% heat-inactivated sheep serum, 0.1%Triton X-100), and then incubated overnight at 4° C. in primaryantibodies against Ki-67 (Santa Cruz, Dallas, Tex.) Fluorescent AlexaFluor 488-conjugated secondary antibodies (Thermo Scientific, Rockford,Ill.) were used for detection. The ki-67 staining intensity wassemi-quantified as—for negative staining, ± for very weak staining, +for weak staining and ++ for strong staining.

9. Microarray Preparation and Analysis

To investigate regulatory role of piRACC on whole-genome mRNAs, HCT116cells were treated with or without piRACC antisense, and subsequentlyperformed Affymetrix GeneChip Human gene 2.0 ST array. The microarrayprobe intensity values (CEL files) were background-corrected andnormalized by Robust Multiarray Average (RMA) method. Comparisonanalysis was performed by using limma package to assess thedifferentiation expression of mRNAs. The genes with ≥1.5 fold change andP-value≤0.01 were selected as differentially expressed. GO analysis wasperformed in DAVID for the differentially expressed genes to evaluatethe enrichment of certain functions. To get insights into piRACC relateddisease and function networks, QIAGEN's Ingenuity Pathway Analysis (seefor example, qiagen.com/ingenuity on the world wide web) was performed.

10. Targets Prediction

To predict the potential targets of piRACC, Miranda v3.3a and RNA22 wasused to search for targets against all human transcripts.

11. Statistical Analysis

All statistical analyses were performed using GraphPad Prism version 6.0or Medcalc version 12.3 programs. Data were expressed as Mean±SD.Statistical differences between groups were determined by Wilcoxon'ssigned rank test, the χ2 test or Mann-Whitney U test. Kaplan-Meieranalysis and log-rank test was used to estimate and compare survivalrates of CRC patients with high and low piRACC expression. ROC curveswere established to determine the cutoff values to discriminate patientswith or without death. The Cox's proportional hazards models were usedto estimate hazard ratios (HRs) for death. All P values were 2-sided,and those less than 0.05 were considered statistically significant.

B. Results

1. Identification of Cancer-Related piRNAs in CRC

The inventors found the mRNA and protein level of PIWIL1 and PIWIL4, twomajor PIWI protein members, were significantly overexpressed in CRCtissues compared to normal tissues, suggesting piRNAs may bedysregulated and involved in CRC development (Table 3 and FIG. 57).Therefore, small RNA-seq analysis was performed to identifyCRC-associated piRNA. The small RNA sequencing libraries were preparedfrom 4 cancer tissues and paired normal mucosa (NM) specimens. Theresults showed that 4 piRNAs (DQ593356, DQ596309, DQ593752 and DQ570994)were differentially expressed between cancer and normal tissues (with ≥2fold change and P value ≤0.01). The expression level of these piRNAs wassubsequently validated in a subset of 20 cancer and paired NM specimensfrom the same cohort. However, similar results were only obtained fromDQ570994 and DQ596309 (FIG. 51A). Due to cohort difference and tumorheterogeneity, the expression level of DQ570994 and DQ596309 was furtherconfirmed in 2 additional independent cohorts. Although no significantdifferences in the expression of DQ596309 was observed, DQ570994 wasconsistently higher in cancer versus normal tissues, with a 5.49-foldincrease (P<0.01) and 7.0 fold change (P<0.01) in each cohort,respectively (FIG. 51B), suggesting piRNA DQ570994 was a potentialonco-piRNA in colorectal cancer. The inventors thus named this piRNA aspiRACC, an abbreviation of piRNA DQ570994 associated with colorectalcancer.

TABLE 3 mRNA level (Oncomine-TCGA) Colon Rectal adeno- adeno- Proteinlevel (Protein Atlas) carcinoma carcinoma Normal vs Normal vs NormalCancer Rectal Colon PIWIL1 Fold 2.465 2.240 9/12 high Low in rectalChange P 4.12 × 10⁻⁹ 7.18 × 10⁻⁸ and colon PIWIL2 Fold 1.190 1.267 NA NAChange P 0.068 0.223 PIWIL3 Fold 1.005 1.046 NA NA Change P 0.465 0.375PIWIL4 Fold 1.523 1.691 3/12 high; Medium in Change Rectal; P 1.47 ×10⁻⁶ 3.26 × 10⁻⁸ 2/12 medium low in colon

In particular, it was noticed that piRACC was not only overexpressed inCRC but also exhibited pan-cancer pattern expression. In the TCGAdatasets, it was found that the expression of piRACC was upregulated invarious type of cancer such as lung, breast, stomach, bladder, kidneyand prostate cancer, highlighting its important key role incarcinogenesis (FIG. 52A-B).

2. Strong Expression of piRACC Correlates with Known RiskClinicopathological Factors of CRC

The inventors next examined the expression patterns of piRACC withregard to their clinical significance in testing cohort (n=195). Theoverexpression of piRACC occurred in a stage dependent manner (P=0.006,Table 1). The distal colon or rectal tumor showed higher expressionlevel of piRACC compared to proximal tumor (P=0.0123). Furthermore,higher expression of piRACC was found in cancer tissues with poordifferentiation (P=0.0566), advanced T stage (P=0.0008), lymph nodemetastasis (P=0.025) and distant metastasis (P=0.0319), suggesting thatpiRACC plays a crucial role in the cancer development.

To further validate the correlation between piRACC expression andclinicopathological variables, the inventors interrogated theseassociations in an additional cohort (n=189). The inventors were able tosuccessfully validate that the upregulated piRACC is associated withadvanced T-stage (P=0.0434), lymph node (P=0.0025) and distantmetastasis (P=0.0027). Collectively, the analyses showed evidence thatexpression of piRACC is overabundant in tumors with high riskclinicopathological features.

3. High Expression of piRACC Associates with Poor Prognosis inColorectal Cancer Patients

To interrogate the impact of piRACC expression on prognosis in CRCpatients, piRACC expression pattern were analyzed in three differentcohorts of a combined total of 771 CRC patients from the TCGA datasets,clinical testing cohort and validation cohort. In the TCGA dataset,piRACC-high expression group showed a strong tendency to be associatedwith poor OS (P=0.0802, HR=1.604; FIG. 52C). Therefore, the prognosticpotency of piRACC was examined in a testing cohort of high qualitytissues with complete follow-up clinical data. As expected, piRACC-highexpression group significantly correlated with poor OS (P=0.0026,HR=2.387; FIG. 52D), suggesting piRACC could be used as prognosticbiomarker for CRC patients. To further confirm the prognostic of piRACCin CRC patient survival, this association was investigated in anadditional cohort. In agreement with other results, piRACC-highexpression groups demonstrated shorter OS (P=0.0002, HR=3.208; FIG.52D), highlighting its clinical relevance as independent prognosticbiomarker in CRC patients. Furthermore, multivariate cox's regressionanalysis revealed that high piRACC expression was an independentpredictor for poor prognosis in both clinical testing and validationcohort (HR: 1.965, 95% CI: 1.0683 to 3.6144, P=0.0298, HR: 2.9347, 95%CI: 1.4584 to 5.9057, P=0.0025, respectively, Table 4). Taken together,these findings elucidate that overexpression of piRACC has clinicalsignificance, and can serve as potential prognostic biomarker in CRCpatients.

TABLE 4 Univariate and multivariate analysis for predictors of overallsurvival in testing and validation cohort Univariate survival analysisMultivariate survival analysis HR 95% CI P HR 95% CI P Testing cohortGender(Male) 0.8471 0.4948-1.4500 0.545 Age(>69) 1.9063 1.0970-3.3125*0.0221 Tumor location 2.3263 1.3379-4.0449 **0.0028 (Proximal)Histological type 2.1597 1.0165-4.5887 *0.0452 1.945 0.9003-4.20190.0905 (Poor) T classification (pT4) 2.6736 1.2045-5.9346 *0.0156 2.15010.9431-4.9017 0.0687 Node involvement 1.6608 0.9700-2.8434 0.0644 1.36420.7813-2.3820 0.2748 (Present) Distant metastasis 4.8339  2.0511-11.3923**0.0003 4.796  1.9696-11.6786 **0.0006 (Present) piRACC expression2.387 1.3300-4.2838 **0.0035 1.965 1.0683-3.6144 *0.0298 level (High)Validation cohort Gender(Male) 1.1471 0.7592-1.7334 0.5145 Age(>69)1.077 0.6065-1.9124 0.8002 Tumor location 0.7355 0.3972-1.3619 0.3284(Proximal) Histological type 3.7535 1.4754-9.5487 **0.0055 3.49141.2996-9.3799 *0.0132 (Poor) T classification (pT4) 3.61 2.0014-6.5114**<0.0001 2.2202 1.1551-4.2673 *0.0167 Node involvement 1.9211.2479-2.9573 **0.003 1.2908 0.5790-2.8776 0.5326 (Present) Distantmetastasis 8.1136  4.4863-14.6739 **<0.0001 4.7427 2.3622-9.5220**<0.0001 (Present) piRACC expression 3.208 1.6989-6.0578 **0.00032.9347 1.4584-5.9057 **0.0025 level (High) HR: Hazard ratio; *P < 0.05;**P < 0.01.

4. piRACC has Multiple Functional Roles in Colorectal Cancer Cells toPromote Tumor Progression

Since high expression of piRACC indicates aggressive clinical behaviorin CRC patients, it was questioned whether piRACC affects biologicalcharacteristics. Several functional assays were performed to determinephenotypic alterations following overexpression or inhibition of piRACCin colon cancer cell. MTT assay was employed to determine theproliferation rates of colon cancer cells transfected with piRACC oligosor antisense. The results showed inhibition of piRACC had a pronouncedsuppression effect on the proliferation of HCT116 and SW480 cells, andin contrast, overexpression of piRACC enhanced cell proliferation (FIG.53A). Meanwhile, colony formation assays were performed to evaluate theeffect of piRACC on the colony-forming ability of single cells in vitro.As shown in FIG. 53B, inhibition of piRACC in HCT116 and SW480 cellsdemonstrated significantly reduced number of colonies compared tocontrol cells, while up-regulation of piRACC markedly increasedcolonies. In line with above findings, inhibition of piRACCsignificantly reduces the percent of Ki-67 strong positive colon cancercells, suggesting that piRACC functions as a positive regulator of cellsurvival (FIG. 53C).

Since high expression of piRACC is associated with lymph node anddistant metastasis, it was assumed that piRACC may regulate cellmigration and invasion as well. As illustrated in FIG. 53D and,inhibition of piRACC significantly suppressed cell migration andinvasion capabilities of both HCT116 and SW480 cells compared to thecontrol cells.

Resistance of programmed cell death is recognized as one of the cancerhallmarks to contribute to tumor metastasis. Based on the clinical data,it was hypothesized that piRACC also plays a key role in apoptosisresistance in colorectal cancer. In line with the hypothesis, inhibitionof piRACC significantly induces apoptosis in HCT116 and SW480 cells(FIG. 53E). Collectively, the data showed newly discovered piRACC exertoncogenic function in CRC through promotion of cell survival, migrationand invasion as well as suppression of apoptosis.

5. piRACC Affects Multiple Cancer-Related Pathways Involved in CellProliferation, Cell Death and Apoptosis

To address the oncogenic mechanism of piRACC in CRC, the impact ofpiRACC on transcriptomes in CRC cell lines was investigated. HCT116cells were treated with or without piRACC antisense and subsequentlyperformed microarray analysis. It was found that a total of 244 mRNAswere detected to be differentially expressed with fold change≥1.5 andP≤0.01. Notably, 168 genes were found upregulated, while 76 genes weredownregulated in piRACC-inhibited cells compared to control cells.

KEGG pathway analysis showed the up-regulated genes are enriched in p53pathway, MAPK pathway and cancer pathway (FIG. 55C). Strikingly, the top10 GO term enrichment analysis for upregulated genes favored cell deathor apoptosis, cell proliferation, protein metabolic process and protein(FIG. 55D)., while the downregulated genes were enriched with genesrelated to chromatin assembly and catalytic activity (FIG. 58).

In order to get insights into disease and function networks, IngenuityPathway Analysis (IPA) was performed based on the microarray data. Theresults disclosed that activated p53 pathway, which was induced bypiRACC inhibition, led to cell apoptosis, necrosis, cell death, contactgrowth inhibition, senescence of cells, and inhibited cellproliferation, colony formation (FIG. 54C). Furthermore, IPA showed thepiRACC acts as important regulator in cell death and survival (data notshown). Based on these findings, these biological process and molecularfunction could contribute to the development of CRC.

6. Identification of piRACC Target mRNAs in CRC

A growing body of studies showed piRNAs have the capabilities to bind todiverse mRNAs and form specific RNA silencing complexes (pi-RISC),leading to RNA repression via imperfect base-pairing between the twoRNAs. The inventors thereafter searched potential target sites of piRACCfrom the upregulated genes. miRANDA and RNA22 tool was used, applyingstringent thermodynamic parameters and binding energy thresholds, topredict biologically relevant RNA-RNA interactions. It was found thatthere are 9 potential targets complementary to piRACC. The examples ofpiRNA:RNA complementarities identified by this approach are shown inFIG. 55A and FIGS. 59-60. These genes have been reported to be involvedin key cellular processes in CRC, including cell death and survival,cell cycle, DNA replication and repair or cell-cell communication (Table5). The inventors further performed qPCR to confirm the expressionchange of target genes after piRACC overexpression or knockdown inHCT116 and SW480 cells and were able to successively validate thesefindings, highlighting that piRACC serves as a master oncogenicregulator in CRC.

TABLE 5 The function of piRACC targets and their expression in CRC GeneExpression* Function** Process** Component** MXD1 down RNA polymerase IIcore cell proliferation nuclear promoter proximal multicellularchromatin region sequence-specific organism nucleus DNA bindingdevelopment protein binding negative regulation protein dimerization oftranscription activity from RNA transcription cofactor polymerase IIactivity promoter transcription corepressor transcription, DNA- activitytemplated transcription factor activity, sequence- specific DNA bindingtranscriptional repressor activity, RNA polymerase II core promoterproximal region sequence-specific binding DUSP5 down MAP kie MAPKcascade nucleoplasm tyrosine/serine/threonine activation of phosphaeactivity MAPK activity phosphae activity dephosphorylation proteinbinding endoderm protein tyrosine formation phosphae activityinactivation of protein MAPK activity tyrosine/serine/threoninepeptidyl-threonine phosphae activity dephosphorylation proteinpeptidyl-tyrosine tyrosine/serine/threonine dephosphorylation phosphaeactivity protein dephosphorylation BTG1 down enzyme binding cellmigration cytoplasm kie binding negative regulation cytoplasm proteinbinding of cell growth nucleus transcription cofactor negativeregulation nucleus activity of cell proliferation positive regulation ofangiogenesis positive regulation of endothelial cell differentiationpositive regulation of fibroblast apoptotic process positive regulationof myoblast differentiation positive regulation of myoblastdifferentiation regulation of transcription, DNA- templated TP53INP1down antioxnt activity apoptotic process PML body protein bindingautophagic cell autophagosome death cytoplasm autophagosome cytoplasmicassembly vesicle cell cycle arrest cytosol cellular oxnt nucleoplasmdetoxification nucleus cellular response to cellular response to ethanolcellular response to hydroperoxide cellular response to methylmethanesulfonate negative regulation of cell migration negativeregulation of cell proliferation positive regulation of apoptoticsignaling pathway positive regulation of autophagy positive regulationof transcription, DNA-templated regulation of apoptotic processregulation of signal transduction by p53 class mediator response to heatresponse to stress transcription, DNA- templated FAS down identicalprotein binding B cell mediated CD95 death- kie binding immunityinducing protease binding activation of signaling protein bindingcysteine-type complex protein complex binding endopeptse activity apicaldendrite receptor activity involved in apical plasma signal transducerapoptotic process membrane activity activation of cell surface tumornecrosis factor- cysteine-type cytoplasm activated receptor endopeptseactivity cytosol activity involved in death-inducing apoptotic signalingsignaling pathway complex activation-induced external side of cell deathof T cells plasma aging membrane apoptotic process extracellularapoptotic signaling exosome pathway extracellular brain developmentspace cellular response to integral cobalt ion component of cellularresponse to plasma estrogen stimulus membrane cellular response tomembrane raft glucose stimulus neuron cellular response to projectionhydrogen peroxide neuronal cell cellular response to body hydrostaticpressure nucleus cellular response to nucleus hyperoxia perinuclearcellular response to region of hypoxia cytoplasm cellular response toplasma interleukin-1 membrane cellular response to plasma lithium ionmembrane cellular response to plasma mechanical membrane stimuluscellular response to phenylalanine chordate embryonic developmentcircadian rhythm dendrite regeneration extrinsic apoptotic signalingpathway extrinsic apoptotic signaling pathway in absence of ligandextrinsic apoptotic signaling pathway via death domain receptors SESN2down leucine binding DNA damage colocalizes_with oxidoreduce activity,response, signal ATG1/ULK1 acting on peroxide as transduction by p53 kiecomplex acceptor class mediator colocalizes_with protein bindingautophagy GATOR2 sulfiredoxin activity cellular oxnt complex NOTsulfiredoxin detoxification colocalizes_with activity NOT cellular oxntTORC2 detoxification complex cellular oxnt cytoplasm detoxificationcytosol cellular response to mitochondrion amino acid stimuluscolocalizes_with cellular response to nucleotide- leucine activatedprotein cellular response to kie complex oxtive stress nucleus fattyacid beta- oxtion glucose ort mitochondrial DNA metabolic processnegative regulation of TORC1 signaling negative regulation of cellgrowth negative regulation of translation in response to endoplasmicreticulum stress positive regulation of macroautophagy positiveregulation of protein localization to nucleus positive regulation oftranscription from RNA polymerase II promoter in response to oxtivestress protein kie B signaling reactive oxygen species metabolic processregulation of cAMP-dependent protein kie activity regulation ofgluconeogenesis involved in cellular glucose homeosis regulation ofprotein phosphorylation regulation of response to reactive oxygenspecies response to glucose response to insulin positive regulation ofNF-kappaB transcription factor activity positive regulation of cellularprotein metabolic process positive regulation of cholesterol effluxpositive regulation of transcription from RNA polymerase II promoterpositive regulation of transcription from RNA polymerase II promoterprotein ort into nucleus, translocation regulation of NF- kappaB ortinto nucleus UPP1 down uridine phosphorylase UMP salvage cytosolactivity cellular response to glucose starvation nucleobase- containingcompound metabolic process nucleotide catabolic process pyrimidinenucleoside catabolic process pyrimidine nucleoside salvage uridinecatabolic process ATF3 down RNA polymerase II core PERK-mediatedCHOP-ATF3 promoter proximal unfolded protein complex regionsequence-specific response nucleolus DNA binding cellular response tonucleoplasm RNA polymerase II amino acid nucleus regulatory regionstarvation sequence-specific DNA gluconeogenesis binding negativeregulation identical protein binding of ERK1 and ERK2 protein bindingcascade protein negative regulation heterodimerization of transcriptionactivity from RNA protein polymerase II homodimerization promoteractivity positive regulation transcription corepressor ofTRAIL-activated activity apoptotic signaling transcription factorpathway activity, RNA positive regulation polymerase II core of cellproliferation promoter proximal positive regulation regionsequence-specific of transcription binding from RNA transcription factorpolymerase II activity, sequence- promoter specific DNA binding positiveregulation transcription regulatory of transcription region DNA bindingfrom RNA transcription regulatory polymerase II region sequence-specificpromoter in DNA binding response to transcriptional activatorendoplasmic activity, RNA reticulum stress polymerase II regulation oftranscription regulatory transcription from region sequence-specific RNApolymerase II binding promoter in transcriptional repressor response toarsenic- activity, RNA containing polymerase II core substance promoterproximal skeletal muscle cell region sequence-specific differentiationbinding transcription from RNA polymerase II promoter *To compare theexpression of target genes in cancer and normal tissues, Oncominedatabase was analyzed (Rhodes, D. R., et al. ONCOMINE: a cancermicroarray database and integrated data-mining platform. Neoplasia, 20046, 1-6). **The function of target gene was provided by Gene OntologyAnnotation (UniProt-GOA) Database

To further validate the in vitro results that piRACC regulated thosetumor suppressors, the expression correlation between piRACC and itstarget genes in colorectal cancer tissues was investigated. The resultsindicated that the expression of these targets were all negativelyassociated with piRACC expression in CRC (P<0.05; FIG. 56). Moreover,several genes have strong inverse correlation with piRACC includingMXD1, BTG1 and FAS, suggesting their expression level are probablytightly synchronized with piRACC function.

C. Discussion

Colorectal cancer is one of the most common cancers worldwide.Therefore, elucidating the molecular mechanisms underlying CRCprogression is critical for the development of new biomarkers ortreatment for the management of patients with this deadly malignancy.Herein, the inventors, for the first time, report piRACC as a noveloncogenic piRNA in CRC. The inventors have made several novelobservations in this study. First, the inventors have discovered thatpiRACC is frequently overexpressed in CRC tissues from differentcohorts, and this overexpression associated with several known riskcliniciopathological factors. Second, this data revealed that patientswith high expression of piRACC had shorter survival compared to thosewith low level of piRACC, highlighting its applicability as a promisingprognostic biomarker in CRC. Third, this is the first study todemonstrate the biological relevance of this piRACC as a tumor-promotingnoncoding RNA in CRC. Fourth, microarray analysis showed piRACCregulates several key cancer pathways, supporting its oncogenic role inCRC. Finally, the inventors discovered several important tumorsuppressors as direct targets of piRACC, and their expression wereobserved inversely correlated with the expression of piRACC, suggestingpiRACC promotes CRC development through inhibition of these target genesat transcriptional level.

It is believed that there are no previous studies reporting the clinicalsignificance of piRNAs in CRC. In this example, the inventors, for thefirst time, demonstrate that piRNAs are highly expressed in colorectalcancer by small RNA-seq analysis. Notably, piRACC was found to beconsistently overexpressed in colorectal cancer tissues across differentcohorts, highlighting its important role in CRC development.Notwithstanding its overexpression in cancer, it was also found thatpiRACC is a strong disease associated biomarker, whose overexpressioncorrelates with known risk clinicopathlogical features such as tumordepth, tumor differentiation and metastasis. Furthermore, another majorfinding is that piRACC was a robust prognostic biomarker for survivalprediction in CRC patients. These findings may help to understand themechanisms of piRNA in metastasis and progression of CRC, and suggestnovel small RNA molecules as biomarkers or therapeutic targets.

To better understand the clinical value of piRACC in CRC, its biologicalsignificance for its contribution to colorectal carcinogenesis should beconsidered. The functional experiments of this example provideconvincing evidence to support for the associations of piRACC withaggressive clinical phenotype, where piRACC promote CRC cells survival,migration and invasion as well as suppression of apoptosis. Consistentwith this paradigm, the microarray analysis clearly showed that piRACCaffects cancer-related pathways and functions as oncogenic regulator indownstream gene network. Accordingly, these results successfully provedthe assumption, whereby the overexpression of piRACC affects generegulatory network for CRC and results in its aggressive phenotype bothbiologically and clinically.

To further decipher the mechanic role of piRACC in CRC, potentialtargets were identified. By using bioinformatics approach, nine‘functionally relevant’ cancer-related genes were identified.Interestingly, these nine candidates are involved in key tumorsuppressive pathway and inversely correlated with piRACC expression,supporting the oncogenic role of piRACC in CRC. Surprisingly, piRACC wasfound not only binds to exon region but also intro region. Recent studyreported that piRNA is able to bind to pre-mRNA intron and subsequentlyleads to the decay of targeted pre-mRNA through nuclear exosomes,suggesting that piRACC may use similar mechanism to downregulate targetgenes. Furthermore, the inventors observed that piRACC could targets3′UTR, CDS or 5′UTR region via perfect or imperfect base-pairing betweenthe two RNAs, by a mechanism that closely resembles that of natureantisense, siRNA or miRNA. Although a number of possible scenarios couldaccount for the interaction between piRACC and its targets, theinventors clearly demonstrated the expression of these targets wassignificantly changed after gain or loss of piRACC in CRC cell lines.

Taken together, these findings implicate piRACC as a potential modulatorof colorectal carcinogenesis, a function possibly linked to piRACCdependent mRNA degradation of its downstream targets. However, theprecise mechanism for the interaction between piRACC and its targetsmerits further investigation. The inventors believe that, to the best oftheir knowledge, that this study represents the first evidence of piRACCas prognostic biomarkers in CRC. Since piRNAs are abundant in cancertissues, with improving profiling platforms and availability of tumorsamples with extensive clinical annotations, it will be helpful toidentify new CRC related piRNAs, further the understanding of theirmechanistic and prognostic contributions to this disease.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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1. A method for treating a patient determined to have colorectal cancercomprising: administering adjuvant therapy to the patient; wherein thepatient was determined to have one or more of the following:differential expression of one or more miRNA, lncRNA, piRNA, mRNA,protein, or 5hmC DNA-modified gene biomarkers compared to a controlsample, wherein the one or more biomarkers are selected from: i)differential expression of miR-30b, miR-32, miR-33a, miR-34a, miR-101,miR-181b, miR-188, miR-191, miR-193b, miR-195, miR-200, miR-200b,miR-362, miR-409, miR-424, miR-425, miR-429, miR-432, miR-592, miR-744,miR-758, miR-1246, miR-3182, miR-3605, miR-3677, miR-4284, miR-4326,CCAT1, CCAT2; piRNA: DQ596309, DQ570994 (piRACC), DQ595807 (piR61919),piR30652 (DQ570540), and/or piR31111 (DQ570999); ii) differential levelsof 5hmC DNA modification of genes: P2RX4, CRISPLD2, and/or FKBP4; andiii) differential mRNA gene expression and/or protein level of: CD44v6,AMT, C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1, LYZ, MGAT5, MMP1, MMP9, NOS2,PIGR, PRAC1, RPL39L, RCC1, SPAG16, AMT2, and/or ITGBL1.
 2. The method ofclaim 1 wherein: the expression level of lncRNAs: CCAT1 and/or CCAT2were increased compared to the control; the expression level of piRNAs:DQ596309, DQ570994 (piRACC), DQ595807, DQ570540, and/or DQ570999 wereincreased compared to the control; and/or the level of 5hmC of genes:P2RX4, CRISPLD2, and/or FKBP4 is decreased compared to the control. 3.The method of claim 1, wherein the method further comprises measuringthe expression level of the miRNAs in a biological sample from thepatient.
 4. The method of claim 1, wherein the method further comprisescomparing the expression level of the biomarker in the biological samplefrom the patient to the expression level of the same biomarker in acontrol biological sample.
 5. The method of claim 1, wherein the patienthas or is determined to have Stage I, II, III, or IV colorectal cancer.6. The method of claim 1, wherein the expression level is normalized. 7.The method of claim 1, wherein the biological sample from the patient isa sample from a primary colorectal cancer tumor, a blood sample, a serumsample, a biopsy sample, or a plasma sample. 8-10. (canceled)
 11. Themethod of claim 1, wherein the control is the level of expression or thebiomarker or level of 5hmC DNA modification in a control biologicalsample.
 12. (canceled)
 13. The method of claim 1, wherein the control isthe level of expression or the biomarker or level of 5hmC DNAmodification in a non-metastatic colorectal cancer tissue. 14.(canceled)
 15. The method of claim 1, wherein the control is the levelof expression or the biomarker or level of 5hmC DNA modification in abiological sample from a patient with non-metastatic or non-progressivecolorectal cancer.
 16. The method of claim 1, wherein the adjuvanttherapy comprises cetuximab, 5-fluorouracil, oxaliplatin, irinotecan,bevacizumab, panitumumab, afibercept, leucovorin, and/or radiotherapy.17. The method of claim 1, wherein the method further comprises surgicalresection of the primary tumor or metastatic tumor.
 18. The method ofclaim 1, wherein the patient does not have and/or has not been diagnosedwith lymph node metastasis.
 19. The method of claim 1, wherein themethod further comprises calculating a risk score based on theexpression levels of the miRNAs in the biological sample from thepatient.
 20. The method of claim 19, wherein the risk score is comparedto a cut-off value. 21-28. (canceled)
 29. The method of claim 1, whereinthe patient was determined to have one or more risk factors selectedfrom poorly differentiated tissues, increased tumor depth; lymphaticinvasion, and venous invasion. 30-32. (canceled)
 33. The method of claim1, wherein the patient was determined to have increased expression ofCD44v6 compared to a control. 34-44. (canceled)
 45. The method of claim1, wherein the patient was determined to have increased CEA expressioncompared to the expression in a control. 46-106. (canceled)
 107. A kitcomprising nucleic acid probes for detecting the expression level ofdifferentially expressed biomarkers in a biological sample; wherein thedifferentially expressed biomarkers comprise one or more of: miRNAs:miR-30b, miR-32, miR-33a, miR-34a, miR-101, miR-181b, miR-188, miR-191,miR-193b, miR-195, miR-200, miR-200b, miR-362, miR-409, miR-424,miR-425, miR-429, miR-432, miR-592, miR-744, miR-758, miR-1246,miR-3182, miR-3605, miR-3677, miR-4284, and/or miR-4326; lncRNA: CCAT1and/or CCAT2; piRNA: DQ596309, DQ570994 (piRACC), DQ595807, DQ570540,and/or DQ570999; 5hmC DNA modification levels of genes: P2RX4, CRISPLD2,and/or FKBP4; and mRNA gene expression and/or protein level of: CD44v6,AMT, C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1, LYZ, MGAT5, MMP1, MMP9, NOS2,PIGR, PRAC1, RPL39L, RCC1, SPAG16, AMT2, and/or ITGBL1. 108-131.(canceled)
 132. A method for treating colorectal cancer in a patient,the method comprising administering one or more of miR-30b, miR-32,miR-33a, miR-34a, miR-101, miR-181b, miR-188, miR-191, miR-193b,miR-195, miR-200, miR-200b, miR-362, miR-409, miR-424, miR-425, miR-429,miR-432, miR-592, miR-744, miR-758, miR-1246, miR-3182, miR-3605,miR-3677, miR-4284, miR-4326, CCAT1, CCAT2; piRNA: DQ596309, DQ570994(piRACC), DQ595807 (DQ595807), DQ570540 (DQ570540), DQ570999 (DQ570999);CD44v6, AMT, C2CD4A, CYP2B6, DEFA6, FOXA1, GSTT1, LYZ, MGAT5, MMP1,MMP9, NOS2, PIGR, PRAC1, RPL39L, RCC1, SPAG16, AMT2, and/or ITGBL1. orantagonist, agonists, or modifiers thereof.